LOCUS       sequence-379966-        7728 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes SaCas9 and an sgRNA targeting mouse Kcnab2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7728)
  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 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"; 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-sgKcnab2
FEATURES             Location/Qualifiers
     source          1..7728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   53..182
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        206..585
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        586..789
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     740..760
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    complement(826..859)
                     /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(910..943)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(955..1035)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             complement(1042..1089)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(1090..4245)
                     /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(4270..4290)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    4322..4355
                     /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    4406..4439
                     /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(4464..4481)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4470..4677
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4684..4932
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4684..4704
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4855..4874
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4954..5029
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   5043..5183
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5043..5172
                     /label=AAV2 ITR
     rep_origin      5258..5713
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5345..5364)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5555..5576
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5730..5749)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5849..5871
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5909..5927)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5995..6099
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6100..6168,6169..6960)
                     /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(6318..6337)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7131..7719
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7620..7639
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gtcctgcagg
       61 cagctgcgcg ctcgctcgct cactgaggcc gcccgggcgt cgggcgacct ttggtcgccc
      121 ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc
      181 ctgcggcctc tagactcgag gcgttgacat tgattattga ctagttatta atagtaatca
      241 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
      301 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
      361 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
      421 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
      481 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
      541 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
      601 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
      661 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
      721 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
      781 agcagagctc tctggctaac taccggtgct agcgtcgact ccggaataac ttcgtatagg
      841 atactttata cgaagttatg cagaatggta gctggattgt agctgctatt agcaatatga
      901 aacctcttaa taacttcgta tagcatacat tatacgaagt tatggcgcgc cttaagcgta
      961 atctggaaca tcgtatgggt aagcgtaatc tggaacatcg tatgggtaag cgtaatctgg
     1021 aacatcgtat gggtaggatc cctttttctt ttttgcctgg ccggcctttt tcgtggccgc
     1081 cggccttttg ccctttttga tgatctgagg gtgcttctta gatttcactt catacaggtt
     1141 gcccagaatg tctgtgctgt acttcttaat gctctgggtc ttggaggcga ttgtcttaat
     1201 gatcctgggg ggcctcttgt cgttcatgtt ttccaggtac tcgcggtagg tgatgtcgat
     1261 catgttcact tcgatccggt tcagcaggtc gttgttcacg ccgatcactc tatacagctc
     1321 gccgttgatc ttgatcagat cgttgttgta gaaggaggcg ataaactcgg cctggttgct
     1381 gatcttcttc agcttcttag cttcctcata gcacttgcta ttcacttcgt agtagttttc
     1441 ttttttgatc acatccagat tcttcacggt cacgaacttg tacacgccat tgtccaggta
     1501 cacgtcgaat ctgtagggct tcagggacag cttcacgacc ttgtttctgc tgttggggta
     1561 gtcgtcggtg atgtccagat gggcgttcag tttgttgccg taatacttaa tcttcttgat
     1621 cacggggccg ttgtcctttt tggagtactt ggtcaggtag ttcccggttt cctcgtagta
     1681 cttgtacagg ggattcttct cgtcgccgta ctgttccata atcagcttca gtttctggta
     1741 ggtctggggg tcgtggtggt acatcagcag cttttcgggg ctcttgttga tcagcttttt
     1801 cagcttgtca ttgtccttgt cgtacaggcc gttcagattg ttcacgatca gggtgttgcc
     1861 cttgtcgtcc ttccgggtgg agtacagggt gtcgttaatc agctctctat taggcttctt
     1921 gtccacccgg tggctgtact tgtagtcctt gaagtcctta atgtgcttga tctggtgggg
     1981 ggtgatgaag atctctttgt actcctgctc ggtttcgatc tcgggcatgc tctcggcctg
     2041 cttttcctcg aacatctggt tttccatcac ttttttggcc ttgtccagtt tcttccactc
     2101 tttgaagatg aaatcggcgt tggcaatgat cagggcgtcc tcggcgtggt gcttgtaccc
     2161 cttgttccgc tctttcttaa acttccactt ccgccgcaga aagctggtga agccgccatt
     2221 gatggacttc actttcacgt ccaggttgtt cactctgaag tagctccgca gcaggttcat
     2281 caggcctctg gtggcgtatc tggtatccac caggttccgg ttgatgaagt ctttctgcac
     2341 ggagaacctg ttgatgtccc gttcttccag cagatactct ttcttggtct tgctgattct
     2401 gcccttgccc ttggccagat tcaggatgtg cttcttgaag gtttcgtagc tgatcttgct
     2461 gtcgctgctg ctcaggtact ggaatggggt ccggttgccc ttcttgctgt tttcttcctg
     2521 cttcacgagc accttgttgt tgaagctgtt gtcgaaggac acgcttctgg ggatgatgtg
     2581 gtccacctca tagttgaagg ggttgttcag cagatcttcc agagggatgg cttccaggct
     2641 gtacaggcac ttgccttcct gcatgtcgtg cagcttgatc ttctcgatca ggtacttggc
     2701 gttctctttg ccggtggtcc ggatgatttc ctcgatccgc tcgttggtct gccggttccg
     2761 cttctgcatc tcgttgatca ttttctgggc gtccttggag ttcttctcgc gggccagctc
     2821 gataatgatg tcgttgggca ggccgtactt cttgatgatg gcgttgatca ctttgatgct
     2881 ctggatgaag cttctcttca cgacggggct caggatgaag tcgtccacca gggtggtggg
     2941 gatctctttc tgctgggaca ggtccacctt cttgggcacc agcttcagcc ggttgaagat
     3001 agcgatctgg ttgtcgttgg tgtgccacag ctcgtccagg atcaggttga tggccttcag
     3061 gctcaggttg tgggtgccgg tatagccctt cagattagag atctgctcga tctcttcctg
     3121 ggtcagctcg gagttcagat tggtcagttc ttcctggatg tcctcgctgc tctggtagat
     3181 ggtcaggatc ttggcaatct gatccagcag ctcggcgttc tcaataatct ctttccgggc
     3241 ggtaatgtcc ttgatgtcgt ggtacacctt caggttggtg aactcgggct tgccggtgct
     3301 ggtcactctg tagcccttaa tatcctcttc gttcacgagg atttctttgg cgatctgctt
     3361 cagggtgggc ttcttcttct gcttgaacac gttctcgatg atctggaact tctcgtaata
     3421 ttccagcttc tcgttctcgt ccctggtgat cacgagattg ttcaggtcgt tcagggcgtt
     3481 gtacaggtcg gcgttgtagg cgtacttcac gctccgcagt tcctcgggga agtaggtgca
     3541 gtggcccatc agcatctcgt accattcttt gatgtccttc cagccgaagg ggctgccctc
     3601 gccaggtccc tcatagtagg tccgccgggt ttccagcagg tcgatgtagg tgtcgatgaa
     3661 gctctggtcc agctggtggt aggccttctg caccttcagc agctgtttgg cttctttcac
     3721 gtagtcgctg gtcttgaatc tgttgatgct gccccgcact tcgccgtctt tcttcagccg
     3781 ttccagctgc agttcggcca cgtatttctc ttccagggcc ttgctgttcc ggctgatctg
     3841 ctctttggtg gacagctcgt tgccggtgtc ctcttccacc tcgttcacgt tgtgcacgcc
     3901 tcttctcttg gccaggtgca gcagggcggc agagaactct tcctcgctca gcttctggct
     3961 caggcccttc actctggcct cgtaggggtt gatgccgctc agctcgctgt ggtcggtcag
     4021 caggttgtag tcgaacagca gcttcttcac tctctggatt ctatgccgcc tccgccgctt
     4081 cagccttctg gcgcctctct tgctccgcct gccctcgttg ttttccacgt tggcctcttt
     4141 gaacagccgc acgccggcat cgatcacgtc ccgtgtctcg tagtcgatga tgccgtagcc
     4201 cacgctggtg atgccgatgt ccaggcccag gatgtagttc cgcttggctg ctgggactcc
     4261 gtggataccg accttccgct tcttctttgg ggccatgcgg ccgctgcaga ccggtgctag
     4321 cataacttcg tataaagtat cctatacgaa gttatttgcc ttaacccaga aattatcact
     4381 gttattcttt agaatggtgc aaagaataac ttcgtataat gtatgctata cgaagttatg
     4441 aattcctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     4501 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     4561 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     4621 ggtggggcag gacagcaagg gggaggattg ggaagagaat agcaggcatg ctggggaggt
     4681 accgagggcc tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga
     4741 gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt
     4801 agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat
     4861 catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa
     4921 ggacgaaaca ccgaatctgg gcaaatctgg cctgttttag tactctggaa acagaatcta
     4981 ctaaaacaag gcaaaatgcc gtgtttatct cgtcaacttg ttggcgagat ttttgcggcc
     5041 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     5101 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     5161 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     5221 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     5281 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg
     5341 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     5401 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     5461 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     5521 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     5581 acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc
     5641 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     5701 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     5761 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     5821 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     5881 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     5941 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     6001 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     6061 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     6121 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     6181 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     6241 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     6301 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     6361 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     6421 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     6481 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     6541 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     6601 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     6661 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     6721 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     6781 tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg
     6841 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     6901 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     6961 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     7021 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     7081 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     7141 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     7201 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     7261 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     7321 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     7381 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     7441 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     7501 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     7561 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     7621 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     7681 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagc
//
