LOCUS       sequence-243605-        7729 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mutagenesis of Slc17a8.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7729)
  AUTHORS   Hunker AC, Soden ME, Krayushkina D, Heymann G, Awatramani R, Zweifel
            LS
  TITLE     Conditional Single Vector CRISPR/SaCas9 Viruses for Efficient 
            Mutagenesis in the Adult Mouse Nervous System.
  JOURNAL   Cell Rep. 2020 Mar 24;30(12):4303-4316.e6. doi: 
            10.1016/j.celrep.2020.02.092.
  PUBMED    32209486
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: 
            "10.1016/j.celrep.2020.02"; journalName: "Cell Rep"; date: 
            "2020-03-24- 24"; volume: "30"; issue: "12"; pages: "4303-4316.e6"
COMMENT     Sequence Label: pAAV-FLEX-SaCas9-U6-sgSlc17a8
FEATURES             Location/Qualifiers
     source          1..7729
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        3..206
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     157..177
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    complement(243..276)
                     /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(327..360)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(372..452)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             complement(459..506)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(507..3662)
                     /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(3687..3707)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    3739..3772
                     /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    3823..3856
                     /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(3881..3898)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3887..4094
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4101..4349
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4101..4121
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4272..4291
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4372..4447
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4461..4601
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4461..4590
                     /label=AAV2 ITR
     rep_origin      4676..5131
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4763..4782)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4973..4994
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5148..5167)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5267..5289
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5327..5345)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5413..5517
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5518..5586,5587..6378)
                     /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(5736..5755)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6549..7137
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7038..7057
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7199..7328
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        join(7352..7729,1..2)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
       61 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
      121 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
      181 ggtgggaggt ctatataagc agagctctct ggctaactac cggtgctagc gtcgactccg
      241 gaataacttc gtataggata ctttatacga agttatgcag aatggtagct ggattgtagc
      301 tgctattagc aatatgaaac ctcttaataa cttcgtatag catacattat acgaagttat
      361 ggcgcgcctt aagcgtaatc tggaacatcg tatgggtaag cgtaatctgg aacatcgtat
      421 gggtaagcgt aatctggaac atcgtatggg taggatccct ttttcttttt tgcctggccg
      481 gcctttttcg tggccgccgg ccttttgccc tttttgatga tctgagggtg cttcttagat
      541 ttcacttcat acaggttgcc cagaatgtct gtgctgtact tcttaatgct ctgggtcttg
      601 gaggcgattg tcttaatgat cctggggggc ctcttgtcgt tcatgttttc caggtactcg
      661 cggtaggtga tgtcgatcat gttcacttcg atccggttca gcaggtcgtt gttcacgccg
      721 atcactctat acagctcgcc gttgatcttg atcagatcgt tgttgtagaa ggaggcgata
      781 aactcggcct ggttgctgat cttcttcagc ttcttagctt cctcatagca cttgctattc
      841 acttcgtagt agttttcttt tttgatcaca tccagattct tcacggtcac gaacttgtac
      901 acgccattgt ccaggtacac gtcgaatctg tagggcttca gggacagctt cacgaccttg
      961 tttctgctgt tggggtagtc gtcggtgatg tccagatggg cgttcagttt gttgccgtaa
     1021 tacttaatct tcttgatcac ggggccgttg tcctttttgg agtacttggt caggtagttc
     1081 ccggtttcct cgtagtactt gtacagggga ttcttctcgt cgccgtactg ttccataatc
     1141 agcttcagtt tctggtaggt ctgggggtcg tggtggtaca tcagcagctt ttcggggctc
     1201 ttgttgatca gctttttcag cttgtcattg tccttgtcgt acaggccgtt cagattgttc
     1261 acgatcaggg tgttgccctt gtcgtccttc cgggtggagt acagggtgtc gttaatcagc
     1321 tctctattag gcttcttgtc cacccggtgg ctgtacttgt agtccttgaa gtccttaatg
     1381 tgcttgatct ggtggggggt gatgaagatc tctttgtact cctgctcggt ttcgatctcg
     1441 ggcatgctct cggcctgctt ttcctcgaac atctggtttt ccatcacttt tttggccttg
     1501 tccagtttct tccactcttt gaagatgaaa tcggcgttgg caatgatcag ggcgtcctcg
     1561 gcgtggtgct tgtacccctt gttccgctct ttcttaaact tccacttccg ccgcagaaag
     1621 ctggtgaagc cgccattgat ggacttcact ttcacgtcca ggttgttcac tctgaagtag
     1681 ctccgcagca ggttcatcag gcctctggtg gcgtatctgg tatccaccag gttccggttg
     1741 atgaagtctt tctgcacgga gaacctgttg atgtcccgtt cttccagcag atactctttc
     1801 ttggtcttgc tgattctgcc cttgcccttg gccagattca ggatgtgctt cttgaaggtt
     1861 tcgtagctga tcttgctgtc gctgctgctc aggtactgga atggggtccg gttgcccttc
     1921 ttgctgtttt cttcctgctt cacgagcacc ttgttgttga agctgttgtc gaaggacacg
     1981 cttctgggga tgatgtggtc cacctcatag ttgaaggggt tgttcagcag atcttccaga
     2041 gggatggctt ccaggctgta caggcacttg ccttcctgca tgtcgtgcag cttgatcttc
     2101 tcgatcaggt acttggcgtt ctctttgccg gtggtccgga tgatttcctc gatccgctcg
     2161 ttggtctgcc ggttccgctt ctgcatctcg ttgatcattt tctgggcgtc cttggagttc
     2221 ttctcgcggg ccagctcgat aatgatgtcg ttgggcaggc cgtacttctt gatgatggcg
     2281 ttgatcactt tgatgctctg gatgaagctt ctcttcacga cggggctcag gatgaagtcg
     2341 tccaccaggg tggtggggat ctctttctgc tgggacaggt ccaccttctt gggcaccagc
     2401 ttcagccggt tgaagatagc gatctggttg tcgttggtgt gccacagctc gtccaggatc
     2461 aggttgatgg ccttcaggct caggttgtgg gtgccggtat agcccttcag attagagatc
     2521 tgctcgatct cttcctgggt cagctcggag ttcagattgg tcagttcttc ctggatgtcc
     2581 tcgctgctct ggtagatggt caggatcttg gcaatctgat ccagcagctc ggcgttctca
     2641 ataatctctt tccgggcggt aatgtccttg atgtcgtggt acaccttcag gttggtgaac
     2701 tcgggcttgc cggtgctggt cactctgtag cccttaatat cctcttcgtt cacgaggatt
     2761 tctttggcga tctgcttcag ggtgggcttc ttcttctgct tgaacacgtt ctcgatgatc
     2821 tggaacttct cgtaatattc cagcttctcg ttctcgtccc tggtgatcac gagattgttc
     2881 aggtcgttca gggcgttgta caggtcggcg ttgtaggcgt acttcacgct ccgcagttcc
     2941 tcggggaagt aggtgcagtg gcccatcagc atctcgtacc attctttgat gtccttccag
     3001 ccgaaggggc tgccctcgcc aggtccctca tagtaggtcc gccgggtttc cagcaggtcg
     3061 atgtaggtgt cgatgaagct ctggtccagc tggtggtagg ccttctgcac cttcagcagc
     3121 tgtttggctt ctttcacgta gtcgctggtc ttgaatctgt tgatgctgcc ccgcacttcg
     3181 ccgtctttct tcagccgttc cagctgcagt tcggccacgt atttctcttc cagggccttg
     3241 ctgttccggc tgatctgctc tttggtggac agctcgttgc cggtgtcctc ttccacctcg
     3301 ttcacgttgt gcacgcctct tctcttggcc aggtgcagca gggcggcaga gaactcttcc
     3361 tcgctcagct tctggctcag gcccttcact ctggcctcgt aggggttgat gccgctcagc
     3421 tcgctgtggt cggtcagcag gttgtagtcg aacagcagct tcttcactct ctggattcta
     3481 tgccgcctcc gccgcttcag ccttctggcg cctctcttgc tccgcctgcc ctcgttgttt
     3541 tccacgttgg cctctttgaa cagccgcacg ccggcatcga tcacgtcccg tgtctcgtag
     3601 tcgatgatgc cgtagcccac gctggtgatg ccgatgtcca ggcccaggat gtagttccgc
     3661 ttggctgctg ggactccgtg gataccgacc ttccgcttct tctttggggc catgcggccg
     3721 ctgcagaccg gtgctagcat aacttcgtat aaagtatcct atacgaagtt atttgcctta
     3781 acccagaaat tatcactgtt attctttaga atggtgcaaa gaataacttc gtataatgta
     3841 tgctatacga agttatgaat tcctagagct cgctgatcag cctcgactgt gccttctagt
     3901 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     3961 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     4021 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agagaatagc
     4081 aggcatgctg gggaggtacc gagggcctat ttcccatgat tccttcatat ttgcatatac
     4141 gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag
     4201 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
     4261 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
     4321 tatatatctt gtggaaagga cgaaacaccg taccctgcct gccatgggat ggttttagta
     4381 ctctggaaac agaatctact aaaacaaggc aaaatgccgt gtttatctcg tcaacttgtt
     4441 ggcgagattt ttgcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg
     4501 cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
     4561 ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt
     4621 tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg
     4681 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     4741 cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     4801 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     4861 ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg
     4921 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     4981 ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga tttataaggg
     5041 attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     5101 aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct
     5161 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     5221 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     5281 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     5341 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     5401 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5461 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     5521 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     5581 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     5641 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     5701 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     5761 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     5821 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     5881 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     5941 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     6001 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     6061 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     6121 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     6181 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc
     6241 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     6301 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     6361 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     6421 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     6481 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6541 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     6601 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     6661 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     6721 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     6781 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     6841 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     6901 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     6961 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     7021 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     7081 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     7141 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgtcc
     7201 tgcaggcagc tgcgcgctcg ctcgctcact gaggccgccc gggcgtcggg cgacctttgg
     7261 tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag
     7321 gggttcctgc ggcctctaga ctcgaggcgt tgacattgat tattgactag ttattaatag
     7381 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
     7441 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
     7501 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
     7561 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
     7621 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
     7681 gactttccta cttggcagta catctacgta ttagtcatcg ctattacca
//
