LOCUS Exported 8140 bp ds-DNA circular SYN 14-OCT-2019 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pAAV2_SauriCas9_P2A_Puro_U6_BsaI SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8140) AUTHORS . TITLE Direct Submission JOURNAL Exported Wednesday, Nov 6, 2019 from SnapGene Viewer 5.0.4 https://www.snapgene.com FEATURES Location/Qualifiers source 1..8140 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 39..130 /label=AAV2 ITR enhancer 154..533 /label=CMV enhancer promoter 534..737 /label=CMV promoter CDS 768..788 /codon_start=1 /label=SV40 NLS /translation="PKKKRKV" primer_bind complement(790..812) /label=SaCas9-R2 CDS 810..3992 /codon_start=1 /label=SauriCas9-KKH /translation="AQENQQKQNYILGLDIGITSVGYGLIDSKTREVIDAGVRLFPEAD SENNSNRRSKRGARRLKRRRIHRLNRVKDLLADYQMIDLNNVPKSTDPYTIRVKGLREP LTKEEFAIALLHIAKRRGLHNISVSMGDEEQDNELSTKQQLQKNAQQLQDKYVCELQLE RLTNINKVRGEKNRFKTEDFVKEVKQLCETQRQYHNIDDQFIQQYIDLVSTRREYFEGP GNGSPYGWDGDLLKWYEKLMGRCTYFPEELRSVKYAYSADLFNALNDLNNLVVTRDDNP KLEYYEKYHIIENVFKQKKNPTLKQIAKEIGVQDYDIRGYRITKSGKPQFTSFKLYHDL KNIFEQAKYLEDVEMLDEIAKILTIYQDEISIKKALDQLPELLTESEKSQIAQLTGYTG THRLSLKCIHIVIDELWESPENQMEIFTRLNLKPKKVEMSEIDSIPTTLVDEFILSPVV KRAFIQSIKVINAVINRFGLPEDIIIELAREKNSKDRRKFINKLQKQNEATRKKIEQLL AKYGNTNAKYMIEKIKLHDMQEGKCLYSLEAIPLEDLLSNPTHYEVDHIIPRSVSFDNS LNNKVLVKQSENSKKGNRTPYQYLSSNESKISYNQFKQHILNLSKAKDRISKKKRDMLL EERDINKFEVQKEFINRNLVDTRYATRELSNLLKTYFSTHDYAVKVKTINGGFTNHLRK VWDFKKHRNHGYKHHAEDALVIANADFLFKTHKALRRTDKILEQPGLEVNDTTVKVDTE EKYQELFETPKQVKNIKQFRDFKYSHRVDKKPNRKLINDTLYSTREIDGETYVVQTLKD LYAKDNEKVKKLFTERPQKILMYQHDPKTFEKLMTILNQYAEAKNPLAAYYEDKGEYVT KYAKKGNGPAIHKIKYIDKKLGSYLDVSNKYPETQNKLVKLSLKSFRFDIYKCEQGYKM VSIGYLDVLKKDNYYYIPKDKYEAEKQKKKIKESDLFVGSFYKNDLIMYEDELFRVIGV NSDINNLVELNMVDITYKDFCEVNNVTGEKHIKKTIGKRVVLIEKYTTDILGNLYKTPL PKKPQLIFKRGEL" primer_bind 2496..2515 /label=R5-BglII-F primer_bind complement(3152..3189) /label=R5-788-R primer_bind 3171..3189 /label=R5-788-F /note="Gln788Lys" unsure 3171..3173 /label=Gln788Lys primer_bind complement(3705..3744) /label=R5-973-R primer_bind 3726..3747 /label=R5-973-F /note="Tyr973Lys" unsure 3726..3728 /label=Tyr973Lys primer_bind complement(3848..3869) /label=R5-1020-R /note="Arg1020His" primer_bind 3852..3889 /label=R5-1020-F unsure 3867..3869 /label=Arg1020His CDS 3993..4040 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind 3993..4010 /label=SaCas9-F primer_bind 4026..4064 /label=Gibsin-P2A-F misc_feature 4053..4118 /label=P2A misc_feature 4119..4715 /label=Puro /label=Puro(-tga) primer_bind complement(4583..4615) /label=Puro-R primer_bind 4598..4619 /label=Puro-F primer_bind complement(4698..4734) /label=Gibson-puro-R CDS 4716..4742 /codon_start=1 /label=HA /translation="YPYDVPDYA" CDS 4743..4769 /codon_start=1 /label=HA /translation="YPYDVPDYA" CDS 4770..4796 /codon_start=1 /label=HA /translation="YPYDVPDYA" misc_feature 4830..5037 /label=PolyA primer_bind complement(4848..4867) /label=polyA-R promoter 5045..5285 /label=U6 Promoter /note="RNA polymerase III promoter for human U6 snRNA" misc_feature 5314..5394 /label=sgRNA repeat_region 5403..5543 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" rep_origin 5618..6073 /direction=RIGHT /label=f1 ori promoter 6355..6459 /label=AmpR promoter CDS 6460..7320 /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" rep_origin 7491..8079 /direction=RIGHT /label=ori 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 ccggtgccac catggcccca aagaagaagc 781 ggaaggtcgg tatccacgga gtcccagcag cccaggagaa ccagcagaag cagaactaca 841 tcctgggcct ggacatcgga atcaccagcg tcggctacgg actgatcgat agcaagacaa 901 gagaagtgat cgacgccggc gttagactct ttccagaagc tgatagcgag aacaactcca 961 accgcagaag caagcggggc gccagacggt taaaacggag aagaatccac cggctgaacc 1021 gggtcaaaga cctgctcgct gattaccaga tgatcgatct taacaatgtt cctaagagca 1081 ccgaccccta caccatcaga gtgaagggcc tccgggagcc tctgacaaaa gaagaattcg 1141 ccatcgccct cctgcatatc gctaagagaa gaggcctgca caacatcagt gtgtccatgg 1201 gcgacgaaga gcaggacaat gaactgagca ccaagcagca gctgcaaaag aatgcccagc 1261 aactgcagga caagtatgtg tgcgaactgc agttagaacg gctgaccaac atcaacaagg 1321 tcagaggcga gaagaacaga tttaagacag aggactttgt gaaagaagtg aaacagctgt 1381 gcgaaaccca gagacagtac cacaacatcg acgaccaatt catccagcag tacatcgacc 1441 tggtgtctac aagacgggag tacttcgagg gccccggcaa cggctctcca tacggctggg 1501 acggcgacct gctgaagtgg tacgagaagc tgatgggcag atgcacctat ttccccgaag 1561 aactgaggtc cgtgaagtac gcctacagcg ccgacctctt caacgccctg aacgacctga 1621 acaacctcgt tgtgaccagg gatgacaatc caaagcttga gtactacgag aagtaccaca 1681 ttattgagaa cgtgttcaag caaaagaaga atcccacact caaacaaatc gccaaagaga 1741 tcggcgtgca agattacgac atccggggct atagaatcac aaagagcggc aaacctcagt 1801 tcacctcttt taagctgtat cacgacctga agaacatctt cgagcaggcc aaatacctgg 1861 aagatgtgga aatgctggac gagatcgcca agatcctgac catctaccag gatgagatta 1921 gcatcaagaa agccctggac cagctgcccg aactgctgac agagagcgag aaatctcaga 1981 tcgcacagct caccggctat acaggcaccc acagactgag cctgaagtgc atccacattg 2041 tgatcgacga gctgtgggag agccccgaga accagatgga aatctttacc agactgaatc 2101 tgaaacctaa gaaggtggaa atgagcgaga tcgacagcat acccaccacc ctggtcgacg 2161 agttcatcct ctcacctgtg gtgaagcggg ccttcatcca gagcatcaag gtaatcaacg 2221 cagtgatcaa tcggttcggc ctgccagagg acatcatcat cgagctggcc agagaaaaga 2281 atagcaagga tcggagaaag ttcattaaca agctgcagaa acaaaatgag gccacaagaa 2341 agaaaatcga acagctgctg gccaagtacg gcaacaccaa tgccaagtac atgatcgaga 2401 agatcaagct gcacgacatg caggagggca agtgcctgta cagcctggag gctattcctc 2461 tggaagacct gctgagcaac ccgacacact acgaagttga ccacattatc cccagatctg 2521 tgagctttga caacagcctg aacaacaaag tgctggtgaa acaaagcgaa aacagcaaga 2581 agggcaatcg caccccttac cagtacctga gcagcaacga gtctaagatt agctacaacc 2641 agtttaagca gcacatcctg aacctgagca aggccaagga cagaatcagc aagaaaaaaa 2701 gagatatgct gctggaagag agagatatca acaagttcga agtgcagaag gaattcatta 2761 accggaacct ggtggataca cggtacgcca ccagagaact gtctaacctg ctgaagacct 2821 acttcagcac ccatgactac gccgtgaagg tgaagaccat caacggcggc ttcactaacc 2881 acctgaggaa ggtgtgggat ttcaagaagc acagaaacca cggctacaag caccacgccg 2941 aagatgccct ggtgatcgcc aacgccgact tcctgtttaa gacacataag gccctgcgga 3001 gaaccgataa gatcctggaa caacctggcc tggaagtgaa tgatacaacc gtgaaagtgg 3061 acaccgagga aaaataccag gagctgttcg agacacctaa gcaagtgaag aacatcaagc 3121 agttccggga cttcaagtac agccaccgag tggacaagaa gcctaaccgg aagcttatca 3181 acgacacact gtactccacc agagagattg atggcgaaac ctacgtggtg cagaccctta 3241 aggatctgta cgccaaggac aacgagaaag tgaagaagct gttcaccgaa agacctcaga 3301 agatcctgat gtaccagcac gaccctaaga ccttcgagaa actgatgaca atcctgaacc 3361 agtacgctga ggccaagaac cctctggctg cttattacga ggacaaaggc gagtacgtga 3421 ccaagtacgc caagaaaggc aatggacctg ccatccacaa gatcaagtat atcgataaga 3481 agcttggatc ttacctggat gttagcaaca agtatcctga gacacagaac aagcttgtga 3541 agctgtccct gaagagcttt agattcgaca tctacaagtg tgaacagggc tacaagatgg 3601 tgtccatcgg atacctggac gtgctgaaga aagataacta ctactacatc cctaaggaca 3661 agtacgaggc cgagaagcag aaaaagaaga tcaaggaatc tgatcttttt gtgggcagct 3721 tctacaagaa cgacctcatc atgtacgagg atgaactgtt cagagtgata ggagtgaaca 3781 gcgacatcaa caatctggtt gagctaaaca tggtcgacat tacctacaag gacttctgcg 3841 aggtgaacaa cgtgacaggc gagaaacaca tcaaaaagac tatcggcaag cgcgtggtcc 3901 tgatcgagaa gtacaccaca gatattctag gcaacctgta caagactccc ctgcctaaga 3961 agccccagct tatcttcaag cggggagaac tgaaaaggcc ggcggccacg aaaaaggccg 4021 gccaggcaaa aaagaaaaag ggatccgcta gcggcagcgg cgccaccaac ttcagcctgc 4081 tgaagcaggc cggcgacgtg gaggagaacc ccggccccat gaccgagtac aagcccacgg 4141 tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg 4201 ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg 4261 agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg 4321 acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt 4381 tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac 4441 agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg 4501 tcggagtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag 4561 tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga aacctccgcg ccccgcaacc 4621 tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac 4681 cgcgcacctg gtgcatgacc cgcaagcccg gtgcctaccc atacgatgtt ccagattacg 4741 cttacccata cgatgttcca gattacgctt acccatacga tgttccagat tacgcttaag 4801 aattcctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt 4861 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta 4921 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg 4981 ggtggggcag gacagcaagg gggaggattg ggaagagaat agcaggcatg ctggggaggt 5041 acctgagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag 5101 agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg 5161 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta 5221 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa 5281 aggacgaaac accggagacc acggcaggtc tcagttttag tactctggaa acagaatcta 5341 ctaaaacaag gcaaaatgcc gtgtttatct cgtcaacttg ttggcgagat ttttgcggcc 5401 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga 5461 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga 5521 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg 5581 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta 5641 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg 5701 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa 5761 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc 5821 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt 5881 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca 5941 acactcaacc ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc 6001 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta 6061 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc 6121 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca 6181 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg 6241 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat 6301 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga 6361 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa 6421 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt 6481 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg 6541 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg 6601 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg 6661 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag 6721 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca 6781 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg 6841 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc 6901 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg 6961 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg 7021 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac 7081 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg 7141 tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg 7201 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact 7261 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa 7321 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt 7381 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag 7441 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct 7501 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 7561 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg 7621 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct 7681 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc 7741 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg 7801 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 7861 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg 7921 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg 7981 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga 8041 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt 8101 ttacggttcc tggccttttg ctggcctttt gctcacatgt //