LOCUS       Exported                7727 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Mutagenesis of Gria2.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-FLEXfrt-SaCas9-U6-sgGria2
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"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        154..533
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        534..737
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     688..708
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    780..813
                     /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(885..965)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             complement(972..1019)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATRKAGQAKKKK"
     CDS             complement(1020..4175)
                     /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(4200..4220)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4410..4427)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4416..4623
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4630..4870
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     4630..4650
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4801..4820
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4901..4976
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4990..5130
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4990..5119
                     /label=AAV2 ITR
     rep_origin      5205..5660
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5292..5311)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5502..5523
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5677..5696)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5796..5818
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5856..5874)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5942..6046
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6047..6907
                     /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(6265..6284)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7078..7666
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7567..7586
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact agttattaat
      181 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
      241 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
      301 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      361 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      421 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
      481 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
      541 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
      601 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
      661 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
      721 tctatataag cagagctctc tggctaacta ccggtgctag cgtcgacaga attggatccg
      781 aagttcctat tctctagaaa gtataggaac ttccaaggcc tgaagttcct attcttcaaa
      841 aggtatagga acttcgcggc cgcttgatat cttggcgcgc cttaagcgta atctggaaca
      901 tcgtatgggt aagcgtaatc tggaacatcg tatgggtaag cgtaatctgg aacatcgtat
      961 gggtaggatc cctttttctt ttttgcctgg ccggcctttc tcgtggccgc cggccttttg
     1021 ccctttttga tgatctgagg gtgcttctta gatttcactt catacaggtt gcccagaatg
     1081 tctgtgctgt acttcttaat gctctgggtc ttggaggcga ttgtcttaat gatcctgggg
     1141 ggcctcttgt cgttcatgtt ttccaggtac tcgcggtagg tgatgtcgat catgttcact
     1201 tcgatccggt tcagcaggtc gttgttcacg ccgatcactc tatacagctc gccgttgatc
     1261 ttgatcagat cgttgttgta gaaggaggcg ataaactcgg cctggttgct gatcttcttc
     1321 agcttcttag cttcctcata gcacttgcta ttcacttcgt agtagttttc ttttttgatc
     1381 acatccagat tcttcacggt cacgaacttg tacacgccat tgtccaggta cacgtcgaat
     1441 ctgtagggct tcagggacag cttcacgacc ttgtttctgc tgttggggta gtcgtcggtg
     1501 atgtccagat gggcgttcag tttgttgccg taatacttaa tcttcttgat cacggggccg
     1561 ttgtcctttt tggagtactt ggtcaggtag ttcccggttt cctcgtagta cttgtacagg
     1621 ggattcttct cgtcgccgta ctgttccata atcagcttca gtttctggta ggtctggggg
     1681 tcgtggtggt acatcagcag cttttcgggg ctcttgttga tcagcttttt cagcttgtca
     1741 ttgtccttgt cgtacaggcc gttcagattg ttcacgatca gggtgttgcc cttgtcgtcc
     1801 ttccgggtgg agtacagggt gtcgttaatc agctctctat taggcttctt gtccacccgg
     1861 tggctgtact tgtagtcctt gaagtcctta atgtgcttga tctggtgggg ggtgatgaag
     1921 atctctttgt actcctgctc ggtttcgatc tcgggcatgc tctcggcctg cttttcctcg
     1981 aacatctggt tttccatcac ttttttggcc ttgtccagtt tcttccactc tttgaagatg
     2041 aaatcggcgt tggcaatgat cagggcgtcc tcggcgtggt gcttgtaccc cttgttccgc
     2101 tctttcttaa acttccactt ccgccgcaga aagctggtga agccgccatt gatggacttc
     2161 actttcacgt ccaggttgtt cactctgaag tagctccgca gcaggttcat caggcctctg
     2221 gtggcgtatc tggtatccac caggttccgg ttgatgaagt ctttctgcac ggagaacctg
     2281 ttgatgtccc gttcttccag cagatactct ttcttggtct tgctgattct gcccttgccc
     2341 ttggccagat tcaggatgtg cttcttgaag gtttcgtagc tgatcttgct gtcgctgctg
     2401 ctcaggtact ggaatggggt ccggttgccc ttcttgctgt tttcttcctg cttcacgagc
     2461 accttgttgt tgaagctgtt gtcgaaggac acgcttctgg ggatgatgtg gtccacctca
     2521 tagttgaagg ggttgttcag cagatcttcc agagggatgg cttccaggct gtacaggcac
     2581 ttgccttcct gcatgtcgtg cagcttgatc ttctcgatca ggtacttggc gttctctttg
     2641 ccggtggtcc ggatgatttc ctcgatccgc tcgttggtct gccggttccg cttctgcatc
     2701 tcgttgatca ttttctgggc gtccttggag ttcttctcgc gggccagctc gataatgatg
     2761 tcgttgggca ggccgtactt cttgatgatg gcgttgatca ctttgatgct ctggatgaag
     2821 cttctcttca cgacggggct caggatgaag tcgtccacca gggtggtggg gatctctttc
     2881 tgctgggaca ggtccacctt cttgggcacc agcttcagcc ggttgaagat agcgatctgg
     2941 ttgtcgttgg tgtgccacag ctcgtccagg atcaggttga tggccttcag gctcaggttg
     3001 tgggtgccgg tatagccctt cagattagag atctgctcga tctcttcctg ggtcagctcg
     3061 gagttcagat tggtcagttc ttcctggatg tcctcgctgc tctggtagat ggtcaggatc
     3121 ttggcaatct gatccagcag ctcggcgttc tcaataatct ctttccgggc ggtaatgtcc
     3181 ttgatgtcgt ggtacacctt caggttggtg aactcgggct tgccggtgct ggtcactctg
     3241 tagcccttaa tatcctcttc gttcacgagg atttctttgg cgatctgctt cagggtgggc
     3301 ttcttcttct gcttgaacac gttctcgatg atctggaact tctcgtaata ttccagcttc
     3361 tcgttctcgt ccctggtgat cacgagattg ttcaggtcgt tcagggcgtt gtacaggtcg
     3421 gcgttgtagg cgtacttcac gctccgcagt tcctcgggga agtaggtgca gtggcccatc
     3481 agcatctcgt accattcttt gatgtccttc cagccgaagg ggctgccctc gccaggtccc
     3541 tcatagtagg tccgccgggt ttccagcagg tcgatgtagg tgtcgatgaa gctctggtcc
     3601 agctggtggt aggccttctg caccttcagc agctgtttgg cttctttcac gtagtcgctg
     3661 gtcttgaatc tgttgatgct gccccgcact tcgccgtctt tcttcagccg ttccagctgc
     3721 agttcggcca cgtatttctc ttccagggcc ttgctgttcc ggctgatctg ctctttggtg
     3781 gacagctcgt tgccggtgtc ctcttccacc tcgttcacgt tgtgcacgcc tcttctcttg
     3841 gccaggtgca gcagggcggc agagaactct tcctcgctca gcttctggct caggcccttc
     3901 actctggcct cgtaggggtt gatgccgctc agctcgctgt ggtcggtcag caggttgtag
     3961 tcgaacagca gcttcttcac tctctggatt ctatgccgcc tccgccgctt cagccttctg
     4021 gcgcctctct tgctccgcct gccctcgttg ttttccacgt tggcctcttt gaacagccgc
     4081 acgccggcat cgatcacgtc ccgtgtctcg tagtcgatga tgccgtagcc cacgctggtg
     4141 atgccgatgt ccaggcccag gatgtagttc cgcttggctg ctgggactcc gtggataccg
     4201 accttccgct tcttctttgg ggccatgcgg ccgctgcaga ccggtttaga tctttactag
     4261 tttaagcttc aattggaagt tcctattctt tctagagtat aggaacttcc tgcagtaggc
     4321 tagcgaagtt cctattcctt ttgaagtata ggaacttcgt cgaggggggg cccgagctta
     4381 tcgatgaatt cctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     4441 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     4501 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     4561 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg
     4621 ggaggtaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
     4681 gttagagaga taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg
     4741 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
     4801 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
     4861 tggaaaggac gaaacaccgt atttccaagg ggcgctgatc gttttagtac tctggaaaca
     4921 gaatctacta aaacaaggca aaatgccgtg tttatctcgt caacttgttg gcgagatttt
     4981 tgcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc
     5041 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
     5101 tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc
     5161 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg
     5221 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     5281 cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     5341 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     5401 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac
     5461 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     5521 tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat
     5581 ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     5641 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata
     5701 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     5761 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     5821 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     5881 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     5941 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     6001 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     6061 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     6121 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     6181 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     6241 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     6301 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     6361 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     6421 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     6481 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     6541 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     6601 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     6661 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     6721 tggctggttt attgctgata aatctggagc cggtgagcgt ggaagccgcg gtatcattgc
     6781 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     6841 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     6901 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     6961 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     7021 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     7081 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     7141 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     7201 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     7261 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     7321 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     7381 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     7441 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     7501 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     7561 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     7621 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     7681 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt
//
