LOCUS       sequence-379962-        7729 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes SaCas9 and an sgRNA targeting mouse Kcna6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7729)
  AUTHORS   Yee JX, Rastani A, Soden ME
  TITLE     The potassium channel auxiliary subunit Kvbeta2 (Kcnab2) regulates 
            Kv1 channels and dopamine neuron firing.
  JOURNAL   J Neurophysiol. 2022 Jul 1;128(1):62-72. doi: 10.1152/jn.00194.2022.
            Epub 2022 Jun 15.
  PUBMED    35788155
REFERENCE   2  (bases 1 to 7729)
  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"; doi: 
            "10.1152/jn.00194.2022"; journalName: "J Neurophysiol"; date: 
            "2022-07-1- 1"; volume: "128"; issue: "1"; pages: "62-72"
COMMENT     Sequence Label: pAAV-FLEX-SaCas9-U6-sgKcna6
FEATURES             Location/Qualifiers
     source          1..7729
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   61..190
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        214..593
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        594..797
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     748..768
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    complement(834..867)
                     /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(918..951)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(963..1043)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             complement(1050..1097)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(1098..4253)
                     /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(4278..4298)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    4330..4363
                     /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    4414..4447
                     /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(4472..4489)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4478..4685
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4692..4940
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4692..4712
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4863..4882
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4963..5038
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   5052..5192
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5052..5181
                     /label=AAV2 ITR
     rep_origin      5267..5722
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5354..5373)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5564..5585
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5739..5758)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5858..5880
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5918..5936)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6004..6108
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6109..6177,6178..6969)
                     /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(6327..6346)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7140..7728
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7629..7648
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
       61 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
      121 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      181 aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact agttattaat
      241 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
      301 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
      361 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      421 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      481 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
      541 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
      601 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
      661 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
      721 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
      781 tctatataag cagagctctc tggctaacta ccggtgctag cgtcgactcc ggaataactt
      841 cgtataggat actttatacg aagttatgca gaatggtagc tggattgtag ctgctattag
      901 caatatgaaa cctcttaata acttcgtata gcatacatta tacgaagtta tggcgcgcct
      961 taagcgtaat ctggaacatc gtatgggtaa gcgtaatctg gaacatcgta tgggtaagcg
     1021 taatctggaa catcgtatgg gtaggatccc tttttctttt ttgcctggcc ggcctttttc
     1081 gtggccgccg gccttttgcc ctttttgatg atctgagggt gcttcttaga tttcacttca
     1141 tacaggttgc ccagaatgtc tgtgctgtac ttcttaatgc tctgggtctt ggaggcgatt
     1201 gtcttaatga tcctgggggg cctcttgtcg ttcatgtttt ccaggtactc gcggtaggtg
     1261 atgtcgatca tgttcacttc gatccggttc agcaggtcgt tgttcacgcc gatcactcta
     1321 tacagctcgc cgttgatctt gatcagatcg ttgttgtaga aggaggcgat aaactcggcc
     1381 tggttgctga tcttcttcag cttcttagct tcctcatagc acttgctatt cacttcgtag
     1441 tagttttctt ttttgatcac atccagattc ttcacggtca cgaacttgta cacgccattg
     1501 tccaggtaca cgtcgaatct gtagggcttc agggacagct tcacgacctt gtttctgctg
     1561 ttggggtagt cgtcggtgat gtccagatgg gcgttcagtt tgttgccgta atacttaatc
     1621 ttcttgatca cggggccgtt gtcctttttg gagtacttgg tcaggtagtt cccggtttcc
     1681 tcgtagtact tgtacagggg attcttctcg tcgccgtact gttccataat cagcttcagt
     1741 ttctggtagg tctgggggtc gtggtggtac atcagcagct tttcggggct cttgttgatc
     1801 agctttttca gcttgtcatt gtccttgtcg tacaggccgt tcagattgtt cacgatcagg
     1861 gtgttgccct tgtcgtcctt ccgggtggag tacagggtgt cgttaatcag ctctctatta
     1921 ggcttcttgt ccacccggtg gctgtacttg tagtccttga agtccttaat gtgcttgatc
     1981 tggtgggggg tgatgaagat ctctttgtac tcctgctcgg tttcgatctc gggcatgctc
     2041 tcggcctgct tttcctcgaa catctggttt tccatcactt ttttggcctt gtccagtttc
     2101 ttccactctt tgaagatgaa atcggcgttg gcaatgatca gggcgtcctc ggcgtggtgc
     2161 ttgtacccct tgttccgctc tttcttaaac ttccacttcc gccgcagaaa gctggtgaag
     2221 ccgccattga tggacttcac tttcacgtcc aggttgttca ctctgaagta gctccgcagc
     2281 aggttcatca ggcctctggt ggcgtatctg gtatccacca ggttccggtt gatgaagtct
     2341 ttctgcacgg agaacctgtt gatgtcccgt tcttccagca gatactcttt cttggtcttg
     2401 ctgattctgc ccttgccctt ggccagattc aggatgtgct tcttgaaggt ttcgtagctg
     2461 atcttgctgt cgctgctgct caggtactgg aatggggtcc ggttgccctt cttgctgttt
     2521 tcttcctgct tcacgagcac cttgttgttg aagctgttgt cgaaggacac gcttctgggg
     2581 atgatgtggt ccacctcata gttgaagggg ttgttcagca gatcttccag agggatggct
     2641 tccaggctgt acaggcactt gccttcctgc atgtcgtgca gcttgatctt ctcgatcagg
     2701 tacttggcgt tctctttgcc ggtggtccgg atgatttcct cgatccgctc gttggtctgc
     2761 cggttccgct tctgcatctc gttgatcatt ttctgggcgt ccttggagtt cttctcgcgg
     2821 gccagctcga taatgatgtc gttgggcagg ccgtacttct tgatgatggc gttgatcact
     2881 ttgatgctct ggatgaagct tctcttcacg acggggctca ggatgaagtc gtccaccagg
     2941 gtggtgggga tctctttctg ctgggacagg tccaccttct tgggcaccag cttcagccgg
     3001 ttgaagatag cgatctggtt gtcgttggtg tgccacagct cgtccaggat caggttgatg
     3061 gccttcaggc tcaggttgtg ggtgccggta tagcccttca gattagagat ctgctcgatc
     3121 tcttcctggg tcagctcgga gttcagattg gtcagttctt cctggatgtc ctcgctgctc
     3181 tggtagatgg tcaggatctt ggcaatctga tccagcagct cggcgttctc aataatctct
     3241 ttccgggcgg taatgtcctt gatgtcgtgg tacaccttca ggttggtgaa ctcgggcttg
     3301 ccggtgctgg tcactctgta gcccttaata tcctcttcgt tcacgaggat ttctttggcg
     3361 atctgcttca gggtgggctt cttcttctgc ttgaacacgt tctcgatgat ctggaacttc
     3421 tcgtaatatt ccagcttctc gttctcgtcc ctggtgatca cgagattgtt caggtcgttc
     3481 agggcgttgt acaggtcggc gttgtaggcg tacttcacgc tccgcagttc ctcggggaag
     3541 taggtgcagt ggcccatcag catctcgtac cattctttga tgtccttcca gccgaagggg
     3601 ctgccctcgc caggtccctc atagtaggtc cgccgggttt ccagcaggtc gatgtaggtg
     3661 tcgatgaagc tctggtccag ctggtggtag gccttctgca ccttcagcag ctgtttggct
     3721 tctttcacgt agtcgctggt cttgaatctg ttgatgctgc cccgcacttc gccgtctttc
     3781 ttcagccgtt ccagctgcag ttcggccacg tatttctctt ccagggcctt gctgttccgg
     3841 ctgatctgct ctttggtgga cagctcgttg ccggtgtcct cttccacctc gttcacgttg
     3901 tgcacgcctc ttctcttggc caggtgcagc agggcggcag agaactcttc ctcgctcagc
     3961 ttctggctca ggcccttcac tctggcctcg taggggttga tgccgctcag ctcgctgtgg
     4021 tcggtcagca ggttgtagtc gaacagcagc ttcttcactc tctggattct atgccgcctc
     4081 cgccgcttca gccttctggc gcctctcttg ctccgcctgc cctcgttgtt ttccacgttg
     4141 gcctctttga acagccgcac gccggcatcg atcacgtccc gtgtctcgta gtcgatgatg
     4201 ccgtagccca cgctggtgat gccgatgtcc aggcccagga tgtagttccg cttggctgct
     4261 gggactccgt ggataccgac cttccgcttc ttctttgggg ccatgcggcc gctgcagacc
     4321 ggtgctagca taacttcgta taaagtatcc tatacgaagt tatttgcctt aacccagaaa
     4381 ttatcactgt tattctttag aatggtgcaa agaataactt cgtataatgt atgctatacg
     4441 aagttatgaa ttcctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca
     4501 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     4561 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     4621 gggggtgggg tggggcagga cagcaagggg gaggattggg aagagaatag caggcatgct
     4681 ggggaggtac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
     4741 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
     4801 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
     4861 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
     4921 tgtggaaagg acgaaacacc gcagcgtgtc agggaacagc gagttttagt actctggaaa
     4981 cagaatctac taaaacaagg caaaatgccg tgtttatctc gtcaacttgt tggcgagatt
     5041 tttgcggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     5101 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc
     5161 agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac
     5221 gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc
     5281 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     5341 gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     5401 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     5461 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag
     5521 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     5581 actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg
     5641 atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     5701 aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca
     5761 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     5821 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     5881 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta
     5941 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     6001 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     6061 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     6121 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     6181 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     6241 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     6301 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     6361 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     6421 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     6481 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     6541 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     6601 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     6661 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     6721 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     6781 gctggctggt ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt
     6841 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     6901 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     6961 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     7021 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     7081 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     7141 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     7201 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     7261 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     7321 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     7381 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     7441 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     7501 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     7561 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     7621 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     7681 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaa
//
