LOCUS Exported 7729 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mutagenesis of Gabrg1. ACCESSION . VERSION . KEYWORDS pAAV-FLEX-SaCas9-U6-sgGabrg1 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 May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7729 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 24..403 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 404..607 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 558..578 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" protein_bind 644..677 /label=lox2272 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GGATACTT). lox2272 sites are compatible with each other, but incompatible with loxP or loxN sites (Lee and Saito, 1988)." protein_bind 728..761 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS complement(773..853) /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA" CDS complement(860..907) /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS complement(908..4063) /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(4088..4108) /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" protein_bind complement(4140..4173) /label=lox2272 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GGATACTT). lox2272 sites are compatible with each other, but incompatible with loxP or loxN sites (Lee and Saito, 1988)." protein_bind complement(4224..4257) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(4282..4299) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 4288..4495 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 4502..4742 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 4502..4522 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 4673..4692 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 4773..4848 /label=Sa gRNA scaffold /note="guide RNA scaffold for the Staphylococcus aureus CRISPR/Cas9 system" repeat_region 4862..5002 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4862..4991 /label=AAV2 ITR rep_origin 5077..5532 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5164..5183) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5374..5395 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5549..5568) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5668..5690 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5728..5746) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5814..5918 /gene="bla" /label=AmpR promoter CDS 5919..6779 /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(6137..6156) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6950..7538 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7439..7458 /label=pBR322ori-F /note="pBR322 origin, forward primer" repeat_region 7600..7729 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" ORIGIN 1 gcggcctcta gactcgaggc gttgacattg attattgact agttattaat agtaatcaat 61 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 121 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 181 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta 241 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt 301 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc 361 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca 421 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat 481 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa 541 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag 601 cagagctctc tggctaacta ccggtgctag cgtcgactcc ggaataactt cgtataggat 661 actttatacg aagttatgca gaatggtagc tggattgtag ctgctattag caatatgaaa 721 cctcttaata acttcgtata gcatacatta tacgaagtta tggcgcgcct taagcgtaat 781 ctggaacatc gtatgggtaa gcgtaatctg gaacatcgta tgggtaagcg taatctggaa 841 catcgtatgg gtaggatccc tttttctttt ttgcctggcc ggcctttttc gtggccgccg 901 gccttttgcc ctttttgatg atctgagggt gcttcttaga tttcacttca tacaggttgc 961 ccagaatgtc tgtgctgtac ttcttaatgc tctgggtctt ggaggcgatt gtcttaatga 1021 tcctgggggg cctcttgtcg ttcatgtttt ccaggtactc gcggtaggtg atgtcgatca 1081 tgttcacttc gatccggttc agcaggtcgt tgttcacgcc gatcactcta tacagctcgc 1141 cgttgatctt gatcagatcg ttgttgtaga aggaggcgat aaactcggcc tggttgctga 1201 tcttcttcag cttcttagct tcctcatagc acttgctatt cacttcgtag tagttttctt 1261 ttttgatcac atccagattc ttcacggtca cgaacttgta cacgccattg tccaggtaca 1321 cgtcgaatct gtagggcttc agggacagct tcacgacctt gtttctgctg ttggggtagt 1381 cgtcggtgat gtccagatgg gcgttcagtt tgttgccgta atacttaatc ttcttgatca 1441 cggggccgtt gtcctttttg gagtacttgg tcaggtagtt cccggtttcc tcgtagtact 1501 tgtacagggg attcttctcg tcgccgtact gttccataat cagcttcagt ttctggtagg 1561 tctgggggtc gtggtggtac atcagcagct tttcggggct cttgttgatc agctttttca 1621 gcttgtcatt gtccttgtcg tacaggccgt tcagattgtt cacgatcagg gtgttgccct 1681 tgtcgtcctt ccgggtggag tacagggtgt cgttaatcag ctctctatta ggcttcttgt 1741 ccacccggtg gctgtacttg tagtccttga agtccttaat gtgcttgatc tggtgggggg 1801 tgatgaagat ctctttgtac tcctgctcgg tttcgatctc gggcatgctc tcggcctgct 1861 tttcctcgaa catctggttt tccatcactt ttttggcctt gtccagtttc ttccactctt 1921 tgaagatgaa atcggcgttg gcaatgatca gggcgtcctc ggcgtggtgc ttgtacccct 1981 tgttccgctc tttcttaaac ttccacttcc gccgcagaaa gctggtgaag ccgccattga 2041 tggacttcac tttcacgtcc aggttgttca ctctgaagta gctccgcagc aggttcatca 2101 ggcctctggt ggcgtatctg gtatccacca ggttccggtt gatgaagtct ttctgcacgg 2161 agaacctgtt gatgtcccgt tcttccagca gatactcttt cttggtcttg ctgattctgc 2221 ccttgccctt ggccagattc aggatgtgct tcttgaaggt ttcgtagctg atcttgctgt 2281 cgctgctgct caggtactgg aatggggtcc ggttgccctt cttgctgttt tcttcctgct 2341 tcacgagcac cttgttgttg aagctgttgt cgaaggacac gcttctgggg atgatgtggt 2401 ccacctcata gttgaagggg ttgttcagca gatcttccag agggatggct tccaggctgt 2461 acaggcactt gccttcctgc atgtcgtgca gcttgatctt ctcgatcagg tacttggcgt 2521 tctctttgcc ggtggtccgg atgatttcct cgatccgctc gttggtctgc cggttccgct 2581 tctgcatctc gttgatcatt ttctgggcgt ccttggagtt cttctcgcgg gccagctcga 2641 taatgatgtc gttgggcagg ccgtacttct tgatgatggc gttgatcact ttgatgctct 2701 ggatgaagct tctcttcacg acggggctca ggatgaagtc gtccaccagg gtggtgggga 2761 tctctttctg ctgggacagg tccaccttct tgggcaccag cttcagccgg ttgaagatag 2821 cgatctggtt gtcgttggtg tgccacagct cgtccaggat caggttgatg gccttcaggc 2881 tcaggttgtg ggtgccggta tagcccttca gattagagat ctgctcgatc tcttcctggg 2941 tcagctcgga gttcagattg gtcagttctt cctggatgtc ctcgctgctc tggtagatgg 3001 tcaggatctt ggcaatctga tccagcagct cggcgttctc aataatctct ttccgggcgg 3061 taatgtcctt gatgtcgtgg tacaccttca ggttggtgaa ctcgggcttg ccggtgctgg 3121 tcactctgta gcccttaata tcctcttcgt tcacgaggat ttctttggcg atctgcttca 3181 gggtgggctt cttcttctgc ttgaacacgt tctcgatgat ctggaacttc tcgtaatatt 3241 ccagcttctc gttctcgtcc ctggtgatca cgagattgtt caggtcgttc agggcgttgt 3301 acaggtcggc gttgtaggcg tacttcacgc tccgcagttc ctcggggaag taggtgcagt 3361 ggcccatcag catctcgtac cattctttga tgtccttcca gccgaagggg ctgccctcgc 3421 caggtccctc atagtaggtc cgccgggttt ccagcaggtc gatgtaggtg tcgatgaagc 3481 tctggtccag ctggtggtag gccttctgca ccttcagcag ctgtttggct tctttcacgt 3541 agtcgctggt cttgaatctg ttgatgctgc cccgcacttc gccgtctttc ttcagccgtt 3601 ccagctgcag ttcggccacg tatttctctt ccagggcctt gctgttccgg ctgatctgct 3661 ctttggtgga cagctcgttg ccggtgtcct cttccacctc gttcacgttg tgcacgcctc 3721 ttctcttggc caggtgcagc agggcggcag agaactcttc ctcgctcagc ttctggctca 3781 ggcccttcac tctggcctcg taggggttga tgccgctcag ctcgctgtgg tcggtcagca 3841 ggttgtagtc gaacagcagc ttcttcactc tctggattct atgccgcctc cgccgcttca 3901 gccttctggc gcctctcttg ctccgcctgc cctcgttgtt ttccacgttg gcctctttga 3961 acagccgcac gccggcatcg atcacgtccc gtgtctcgta gtcgatgatg ccgtagccca 4021 cgctggtgat gccgatgtcc aggcccagga tgtagttccg cttggctgct gggactccgt 4081 ggataccgac cttccgcttc ttctttgggg ccatgcggcc gctgcagacc ggtgctagca 4141 taacttcgta taaagtatcc tatacgaagt tatttgcctt aacccagaaa ttatcactgt 4201 tattctttag aatggtgcaa agaataactt cgtataatgt atgctatacg aagttatgaa 4261 ttcctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt 4321 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat 4381 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg 4441 tggggcagga cagcaagggg gaggattggg aagagaatag caggcatgct ggggaggtac 4501 cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga 4561 gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag 4621 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca 4681 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg 4741 acgaaacacc gtctcctgtg gagtcaaact aggttttagt actctggaaa cagaatctac 4801 taaaacaagg caaaatgccg tgtttatctc gtcaacttgt tggcgagatt tttgcggccg 4861 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag 4921 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag 4981 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc 5041 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa 5101 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc 5161 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag 5221 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca 5281 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc 5341 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa 5401 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct 5461 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa 5521 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc 5581 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat 5641 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt 5701 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg 5761 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa 5821 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 5881 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg 5941 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc 6001 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg 6061 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga 6121 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc 6181 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag 6241 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga 6301 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg 6361 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 6421 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt 6481 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact 6541 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt 6601 ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg 6661 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta 6721 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac 6781 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta 6841 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt 6901 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt 6961 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 7021 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc 7081 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg 7141 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg 7201 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt 7261 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac 7321 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg 7381 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg 7441 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat 7501 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt 7561 tacggttcct ggccttttgc tggccttttg ctcacatgtc ctgcaggcag ctgcgcgctc 7621 gctcgctcac tgaggccgcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc 7681 gagcgagcgc gcagagaggg agtggccaac tccatcacta ggggttcct //