LOCUS       sequence-379970-        7728 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes SaCas9 and an sgRNA targeting mouse Kcna2.
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-sgKcna2
FEATURES             Location/Qualifiers
     source          1..7728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   42..171
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        195..574
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        575..778
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     729..749
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    complement(815..848)
                     /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(899..932)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(944..1024)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             complement(1031..1078)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(1079..4234)
                     /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(4259..4279)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    4311..4344
                     /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    4395..4428
                     /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(4453..4470)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4459..4666
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4673..4921
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4673..4693
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4844..4863
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4943..5018
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   5032..5172
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5032..5161
                     /label=AAV2 ITR
     rep_origin      5247..5702
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5334..5353)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5544..5565
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5719..5738)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5838..5860
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5898..5916)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5984..6088
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6089..6157,6158..6949)
                     /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(6307..6326)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7120..7708
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7609..7628
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tttacggttc ctggcctttt gctggccttt tgctcacatg tcctgcaggc agctgcgcgc
       61 tcgctcgctc actgaggccg cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga
      121 gcgagcgagc gcgcagagag ggagtggcca actccatcac taggggttcc tgcggcctct
      181 agactcgagg cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
      241 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
      301 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
      361 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
      421 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
      481 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
      541 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
      601 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
      661 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
      721 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct
      781 ctggctaact accggtgcta gcgtcgactc cggaataact tcgtatagga tactttatac
      841 gaagttatgc agaatggtag ctggattgta gctgctatta gcaatatgaa acctcttaat
      901 aacttcgtat agcatacatt atacgaagtt atggcgcgcc ttaagcgtaa tctggaacat
      961 cgtatgggta agcgtaatct ggaacatcgt atgggtaagc gtaatctgga acatcgtatg
     1021 ggtaggatcc ctttttcttt tttgcctggc cggccttttt cgtggccgcc ggccttttgc
     1081 cctttttgat gatctgaggg tgcttcttag atttcacttc atacaggttg cccagaatgt
     1141 ctgtgctgta cttcttaatg ctctgggtct tggaggcgat tgtcttaatg atcctggggg
     1201 gcctcttgtc gttcatgttt tccaggtact cgcggtaggt gatgtcgatc atgttcactt
     1261 cgatccggtt cagcaggtcg ttgttcacgc cgatcactct atacagctcg ccgttgatct
     1321 tgatcagatc gttgttgtag aaggaggcga taaactcggc ctggttgctg atcttcttca
     1381 gcttcttagc ttcctcatag cacttgctat tcacttcgta gtagttttct tttttgatca
     1441 catccagatt cttcacggtc acgaacttgt acacgccatt gtccaggtac acgtcgaatc
     1501 tgtagggctt cagggacagc ttcacgacct tgtttctgct gttggggtag tcgtcggtga
     1561 tgtccagatg ggcgttcagt ttgttgccgt aatacttaat cttcttgatc acggggccgt
     1621 tgtccttttt ggagtacttg gtcaggtagt tcccggtttc ctcgtagtac ttgtacaggg
     1681 gattcttctc gtcgccgtac tgttccataa tcagcttcag tttctggtag gtctgggggt
     1741 cgtggtggta catcagcagc ttttcggggc tcttgttgat cagctttttc agcttgtcat
     1801 tgtccttgtc gtacaggccg ttcagattgt tcacgatcag ggtgttgccc ttgtcgtcct
     1861 tccgggtgga gtacagggtg tcgttaatca gctctctatt aggcttcttg tccacccggt
     1921 ggctgtactt gtagtccttg aagtccttaa tgtgcttgat ctggtggggg gtgatgaaga
     1981 tctctttgta ctcctgctcg gtttcgatct cgggcatgct ctcggcctgc ttttcctcga
     2041 acatctggtt ttccatcact tttttggcct tgtccagttt cttccactct ttgaagatga
     2101 aatcggcgtt ggcaatgatc agggcgtcct cggcgtggtg cttgtacccc ttgttccgct
     2161 ctttcttaaa cttccacttc cgccgcagaa agctggtgaa gccgccattg atggacttca
     2221 ctttcacgtc caggttgttc actctgaagt agctccgcag caggttcatc aggcctctgg
     2281 tggcgtatct ggtatccacc aggttccggt tgatgaagtc tttctgcacg gagaacctgt
     2341 tgatgtcccg ttcttccagc agatactctt tcttggtctt gctgattctg cccttgccct
     2401 tggccagatt caggatgtgc ttcttgaagg tttcgtagct gatcttgctg tcgctgctgc
     2461 tcaggtactg gaatggggtc cggttgccct tcttgctgtt ttcttcctgc ttcacgagca
     2521 ccttgttgtt gaagctgttg tcgaaggaca cgcttctggg gatgatgtgg tccacctcat
     2581 agttgaaggg gttgttcagc agatcttcca gagggatggc ttccaggctg tacaggcact
     2641 tgccttcctg catgtcgtgc agcttgatct tctcgatcag gtacttggcg ttctctttgc
     2701 cggtggtccg gatgatttcc tcgatccgct cgttggtctg ccggttccgc ttctgcatct
     2761 cgttgatcat tttctgggcg tccttggagt tcttctcgcg ggccagctcg ataatgatgt
     2821 cgttgggcag gccgtacttc ttgatgatgg cgttgatcac tttgatgctc tggatgaagc
     2881 ttctcttcac gacggggctc aggatgaagt cgtccaccag ggtggtgggg atctctttct
     2941 gctgggacag gtccaccttc ttgggcacca gcttcagccg gttgaagata gcgatctggt
     3001 tgtcgttggt gtgccacagc tcgtccagga tcaggttgat ggccttcagg ctcaggttgt
     3061 gggtgccggt atagcccttc agattagaga tctgctcgat ctcttcctgg gtcagctcgg
     3121 agttcagatt ggtcagttct tcctggatgt cctcgctgct ctggtagatg gtcaggatct
     3181 tggcaatctg atccagcagc tcggcgttct caataatctc tttccgggcg gtaatgtcct
     3241 tgatgtcgtg gtacaccttc aggttggtga actcgggctt gccggtgctg gtcactctgt
     3301 agcccttaat atcctcttcg ttcacgagga tttctttggc gatctgcttc agggtgggct
     3361 tcttcttctg cttgaacacg ttctcgatga tctggaactt ctcgtaatat tccagcttct
     3421 cgttctcgtc cctggtgatc acgagattgt tcaggtcgtt cagggcgttg tacaggtcgg
     3481 cgttgtaggc gtacttcacg ctccgcagtt cctcggggaa gtaggtgcag tggcccatca
     3541 gcatctcgta ccattctttg atgtccttcc agccgaaggg gctgccctcg ccaggtccct
     3601 catagtaggt ccgccgggtt tccagcaggt cgatgtaggt gtcgatgaag ctctggtcca
     3661 gctggtggta ggccttctgc accttcagca gctgtttggc ttctttcacg tagtcgctgg
     3721 tcttgaatct gttgatgctg ccccgcactt cgccgtcttt cttcagccgt tccagctgca
     3781 gttcggccac gtatttctct tccagggcct tgctgttccg gctgatctgc tctttggtgg
     3841 acagctcgtt gccggtgtcc tcttccacct cgttcacgtt gtgcacgcct cttctcttgg
     3901 ccaggtgcag cagggcggca gagaactctt cctcgctcag cttctggctc aggcccttca
     3961 ctctggcctc gtaggggttg atgccgctca gctcgctgtg gtcggtcagc aggttgtagt
     4021 cgaacagcag cttcttcact ctctggattc tatgccgcct ccgccgcttc agccttctgg
     4081 cgcctctctt gctccgcctg ccctcgttgt tttccacgtt ggcctctttg aacagccgca
     4141 cgccggcatc gatcacgtcc cgtgtctcgt agtcgatgat gccgtagccc acgctggtga
     4201 tgccgatgtc caggcccagg atgtagttcc gcttggctgc tgggactccg tggataccga
     4261 ccttccgctt cttctttggg gccatgcggc cgctgcagac cggtgctagc ataacttcgt
     4321 ataaagtatc ctatacgaag ttatttgcct taacccagaa attatcactg ttattcttta
     4381 gaatggtgca aagaataact tcgtataatg tatgctatac gaagttatga attcctagag
     4441 ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     4501 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     4561 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     4621 acagcaaggg ggaggattgg gaagagaata gcaggcatgc tggggaggta ccgagggcct
     4681 atttcccatg attccttcat atttgcatat acgatacaag gctgttagag agataattgg
     4741 aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata
     4801 atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac
     4861 cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac
     4921 cgaagcaatg gagatgtttc gggttttagt actctggaaa cagaatctac taaaacaagg
     4981 caaaatgccg tgtttatctc gtcaacttgt tggcgagatt tttgcggccg caggaacccc
     5041 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac
     5101 caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca
     5161 gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     5221 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg
     5281 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt
     5341 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     5401 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     5461 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     5521 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc
     5581 tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct attggttaaa
     5641 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     5701 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     5761 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     5821 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     5881 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     5941 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     6001 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     6061 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     6121 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     6181 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     6241 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     6301 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     6361 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     6421 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     6481 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     6541 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     6601 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     6661 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     6721 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     6781 taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg ggccagatgg
     6841 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     6901 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     6961 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     7021 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     7081 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     7141 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     7201 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     7261 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     7321 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     7381 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     7441 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     7501 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     7561 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     7621 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     7681 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctt
//
