LOCUS       Exported                7727 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Mutagenesis of Slc17a6.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-FLEXfrt-SaCas9-U6-sgSlc17a6
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7727)
  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 7727)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7727
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        132..511
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        512..715
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     666..686
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    758..791
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             complement(863..943)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             complement(950..997)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATRKAGQAKKKK"
     CDS             complement(998..4153)
                     /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(4178..4198)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4388..4405)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4394..4601
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4608..4848
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     4608..4628
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4779..4798
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4879..4954
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4968..5108
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4968..5097
                     /label=AAV2 ITR
     rep_origin      5183..5638
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5270..5289)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5480..5501
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5655..5674)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5774..5796
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5834..5852)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5920..6024
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6025..6885
                     /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(6243..6262)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7056..7644
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7545..7564
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7706..108
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 ctcgctcact gaggccgccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg
       61 agcgagcgcg cagagaggga gtggccaact ccatcactag gggttcctgc ggcctctaga
      121 ctcgaggcgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt
      181 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
      241 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
      301 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
      361 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
      421 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
      481 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
      541 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
      601 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
      661 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg
      721 gctaactacc ggtgctagcg tcgacagaat tggatccgaa gttcctattc tctagaaagt
      781 ataggaactt ccaaggcctg aagttcctat tcttcaaaag gtataggaac ttcgcggccg
      841 cttgatatct tggcgcgcct taagcgtaat ctggaacatc gtatgggtaa gcgtaatctg
      901 gaacatcgta tgggtaagcg taatctggaa catcgtatgg gtaggatccc tttttctttt
      961 ttgcctggcc ggcctttctc gtggccgccg gccttttgcc ctttttgatg atctgagggt
     1021 gcttcttaga tttcacttca tacaggttgc ccagaatgtc tgtgctgtac ttcttaatgc
     1081 tctgggtctt ggaggcgatt gtcttaatga tcctgggggg cctcttgtcg ttcatgtttt
     1141 ccaggtactc gcggtaggtg atgtcgatca tgttcacttc gatccggttc agcaggtcgt
     1201 tgttcacgcc gatcactcta tacagctcgc cgttgatctt gatcagatcg ttgttgtaga
     1261 aggaggcgat aaactcggcc tggttgctga tcttcttcag cttcttagct tcctcatagc
     1321 acttgctatt cacttcgtag tagttttctt ttttgatcac atccagattc ttcacggtca
     1381 cgaacttgta cacgccattg tccaggtaca cgtcgaatct gtagggcttc agggacagct
     1441 tcacgacctt gtttctgctg ttggggtagt cgtcggtgat gtccagatgg gcgttcagtt
     1501 tgttgccgta atacttaatc ttcttgatca cggggccgtt gtcctttttg gagtacttgg
     1561 tcaggtagtt cccggtttcc tcgtagtact tgtacagggg attcttctcg tcgccgtact
     1621 gttccataat cagcttcagt ttctggtagg tctgggggtc gtggtggtac atcagcagct
     1681 tttcggggct cttgttgatc agctttttca gcttgtcatt gtccttgtcg tacaggccgt
     1741 tcagattgtt cacgatcagg gtgttgccct tgtcgtcctt ccgggtggag tacagggtgt
     1801 cgttaatcag ctctctatta ggcttcttgt ccacccggtg gctgtacttg tagtccttga
     1861 agtccttaat gtgcttgatc tggtgggggg tgatgaagat ctctttgtac tcctgctcgg
     1921 tttcgatctc gggcatgctc tcggcctgct tttcctcgaa catctggttt tccatcactt
     1981 ttttggcctt gtccagtttc ttccactctt tgaagatgaa atcggcgttg gcaatgatca
     2041 gggcgtcctc ggcgtggtgc ttgtacccct tgttccgctc tttcttaaac ttccacttcc
     2101 gccgcagaaa gctggtgaag ccgccattga tggacttcac tttcacgtcc aggttgttca
     2161 ctctgaagta gctccgcagc aggttcatca ggcctctggt ggcgtatctg gtatccacca
     2221 ggttccggtt gatgaagtct ttctgcacgg agaacctgtt gatgtcccgt tcttccagca
     2281 gatactcttt cttggtcttg ctgattctgc ccttgccctt ggccagattc aggatgtgct
     2341 tcttgaaggt ttcgtagctg atcttgctgt cgctgctgct caggtactgg aatggggtcc
     2401 ggttgccctt cttgctgttt tcttcctgct tcacgagcac cttgttgttg aagctgttgt
     2461 cgaaggacac gcttctgggg atgatgtggt ccacctcata gttgaagggg ttgttcagca
     2521 gatcttccag agggatggct tccaggctgt acaggcactt gccttcctgc atgtcgtgca
     2581 gcttgatctt ctcgatcagg tacttggcgt tctctttgcc ggtggtccgg atgatttcct
     2641 cgatccgctc gttggtctgc cggttccgct tctgcatctc gttgatcatt ttctgggcgt
     2701 ccttggagtt cttctcgcgg gccagctcga taatgatgtc gttgggcagg ccgtacttct
     2761 tgatgatggc gttgatcact ttgatgctct ggatgaagct tctcttcacg acggggctca
     2821 ggatgaagtc gtccaccagg gtggtgggga tctctttctg ctgggacagg tccaccttct
     2881 tgggcaccag cttcagccgg ttgaagatag cgatctggtt gtcgttggtg tgccacagct
     2941 cgtccaggat caggttgatg gccttcaggc tcaggttgtg ggtgccggta tagcccttca
     3001 gattagagat ctgctcgatc tcttcctggg tcagctcgga gttcagattg gtcagttctt
     3061 cctggatgtc ctcgctgctc tggtagatgg tcaggatctt ggcaatctga tccagcagct
     3121 cggcgttctc aataatctct ttccgggcgg taatgtcctt gatgtcgtgg tacaccttca
     3181 ggttggtgaa ctcgggcttg ccggtgctgg tcactctgta gcccttaata tcctcttcgt
     3241 tcacgaggat ttctttggcg atctgcttca gggtgggctt cttcttctgc ttgaacacgt
     3301 tctcgatgat ctggaacttc tcgtaatatt ccagcttctc gttctcgtcc ctggtgatca
     3361 cgagattgtt caggtcgttc agggcgttgt acaggtcggc gttgtaggcg tacttcacgc
     3421 tccgcagttc ctcggggaag taggtgcagt ggcccatcag catctcgtac cattctttga
     3481 tgtccttcca gccgaagggg ctgccctcgc caggtccctc atagtaggtc cgccgggttt
     3541 ccagcaggtc gatgtaggtg tcgatgaagc tctggtccag ctggtggtag gccttctgca
     3601 ccttcagcag ctgtttggct tctttcacgt agtcgctggt cttgaatctg ttgatgctgc
     3661 cccgcacttc gccgtctttc ttcagccgtt ccagctgcag ttcggccacg tatttctctt
     3721 ccagggcctt gctgttccgg ctgatctgct ctttggtgga cagctcgttg ccggtgtcct
     3781 cttccacctc gttcacgttg tgcacgcctc ttctcttggc caggtgcagc agggcggcag
     3841 agaactcttc ctcgctcagc ttctggctca ggcccttcac tctggcctcg taggggttga
     3901 tgccgctcag ctcgctgtgg tcggtcagca ggttgtagtc gaacagcagc ttcttcactc
     3961 tctggattct atgccgcctc cgccgcttca gccttctggc gcctctcttg ctccgcctgc
     4021 cctcgttgtt ttccacgttg gcctctttga acagccgcac gccggcatcg atcacgtccc
     4081 gtgtctcgta gtcgatgatg ccgtagccca cgctggtgat gccgatgtcc aggcccagga
     4141 tgtagttccg cttggctgct gggactccgt ggataccgac cttccgcttc ttctttgggg
     4201 ccatgcggcc gctgcagacc ggtttagatc tttactagtt taagcttcaa ttggaagttc
     4261 ctattctttc tagagtatag gaacttcctg cagtaggcta gcgaagttcc tattcctttt
     4321 gaagtatagg aacttcgtcg agggggggcc cgagcttatc gatgaattcc tagagctcgc
     4381 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     4441 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     4501 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     4561 aagggggagg attgggaaga gaatagcagg catgctgggg aggtaccgag ggcctatttc
     4621 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attggaatta
     4681 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc
     4741 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa
     4801 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgtat
     4861 gctgatccca tctgcagcgt tttagtactc tggaaacaga atctactaaa acaaggcaaa
     4921 atgccgtgtt tatctcgtca acttgttggc gagatttttg cggccgcagg aacccctagt
     4981 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     5041 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg
     5101 cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     5161 catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     5221 gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct
     5281 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     5341 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg
     5401 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     5461 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc
     5521 tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat
     5581 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta
     5641 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     5701 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     5761 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     5821 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     5881 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     5941 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     6001 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     6061 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     6121 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     6181 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     6241 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     6301 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     6361 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     6421 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     6481 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     6541 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     6601 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     6661 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     6721 tctggagccg gtgagcgtgg aagccgcggt atcattgcag cactggggcc agatggtaag
     6781 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     6841 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     6901 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     6961 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     7021 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     7081 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     7141 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     7201 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     7261 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     7321 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     7381 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     7441 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     7501 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     7561 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     7621 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     7681 ttttgctggc cttttgctca catgtcctgc aggcagctgc gcgctcg
//
