LOCUS Exported 8239 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expresses an spCas9 under the control of a CMV promoter; The spCas9 is flanked by COL4A5 sgRNA targets to induce its self-cleaving and inactivation. ACCESSION . VERSION . KEYWORDS pAAV_COL4A5_self-cleavingCas9 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8239) TITLE New frontiers to cure Alport syndrome: COL4A3 and COL4A5 gene editing in podocyte-lineage cells JOURNAL Unpublished REFERENCE 2 (bases 1 to 8239) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8239 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 107..128 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 143..173 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 181..197 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 186..208 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 205..221 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 205..221 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" repeat_region 248..377 /label=AAV2 ITR primer_bind 457..473 /label=pBluescriptKS /note="For pBluescript vector" primer_bind 458..474 /label=KS primer /note="common sequencing primer, one of multiple similar variants" promoter 616..819 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 770..790 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" CDS 884..949 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 956..976 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1001..5101 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 5102..5149 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5245..5262) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5251..5458 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5546..5675 /label=AAV2 ITR primer_bind complement(5694..5711) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(5694..5710) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5703..5725) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 5852..6307 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5939..5958) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6149..6170 /label=F1ori-F /note="F1 origin, forward primer" promoter 6334..6438 /gene="bla" /label=AmpR promoter CDS 6439..7299 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVVMVTTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(6657..6676) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7470..8058 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7959..7978 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 8212..8229 /label=L4440 /note="L4440 vector, forward primer" ORIGIN 1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccagattta 241 attaaggctg cgcgctcgct cgctcactga ggccgcccgg gcaaagcccg ggcgtcgggc 301 gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc 361 catcactagg ggttccttgt agttaatgat taacccgcca tgctacttat ctacgtagcc 421 atgctctagg aagatcggaa ttcgccctta agctagtcga ggtcgacggt atcgataagc 481 ttgattatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat 541 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc 601 atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt 661 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac 721 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc 781 ggtaggcgtg tacggtggga ggtctatata agcagagctg gtttagtgaa ccgtcagatc 841 cgctagagct tggatcccct ggagggccga aatcaggggg atggactata aggaccacga 901 cggagactac aaggatcatg atattgatta caaagacgat gacgataaga tggccccaaa 961 gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt acagcatcgg 1021 cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc 1081 cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat 1141 cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc 1201 cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa 1261 cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga 1321 agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta 1381 ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa 1441 ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt 1501 cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct 1561 ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc 1621 caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca 1681 gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct 1741 gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa 1801 ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga 1861 cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt 1921 gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca 1981 ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa 2041 agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag 2101 ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga 2161 actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg 2221 cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga 2281 tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat 2341 cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa 2401 gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc 2461 ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct 2521 gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa 2581 atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat 2641 cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta 2701 cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa 2761 cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga 2821 caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga 2881 cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat 2941 gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa 3001 cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt 3061 cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat 3121 ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc 3181 cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt 3241 gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca 3301 gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat 3361 caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa 3421 cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact 3481 ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa 3541 ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga 3601 caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa 3661 cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct 3721 gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac 3781 aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa 3841 gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa 3901 ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta 3961 cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt 4021 cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga 4081 aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac 4141 cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga 4201 aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag 4261 catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga 4321 gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc 4381 taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa 4441 agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat 4501 catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa 4561 agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa 4621 cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct 4681 gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc 4741 ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga 4801 gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga 4861 caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa 4921 tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga 4981 caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat 5041 ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga 5101 caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagt aagaattcct 5161 agagctcgct gaccccctga tttcggccct ccaggccgcg gcctgctgcc ggctctgcgg 5221 cctcttccgc gtcttcgaga tctgcctcga ctgtgccttc tagttgccag ccatctgttg 5281 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct 5341 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg 5401 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggact 5461 cgagttaagg gcgaattccc gattaggatc ttcctagagc atggctacgt agataagtag 5521 catggcgggt taatcattaa ctacaaggaa cccctagtga tggagttggc cactccctct 5581 ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt 5641 gcccgggcgg cctcagtgag cgagcgagcg cgcagcctta attaacctaa ttcactggcc 5701 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca 5761 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc 5821 caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg 5881 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct 5941 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta 6001 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa 6061 cttgattagg gtgatggttc acgtagtggg ccatcgcccc gatagacggt ttttcgccct 6121 ttgacgctgg agttcacgtt cctcaatagt ggactcttgt tccaaactgg aacaacactc 6181 aaccctatct cggtctattc ttttgattta taagggattt ttccgatttc ggcctattgg 6241 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt 6301 ataatttcag gtggcatctt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 6361 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 6421 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt 6481 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct 6541 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaatag tggtaagatc 6601 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 6661 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac 6721 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 6781 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 6841 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 6901 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac 6961 gagcgtgaca ccacgatgcc tgtagtaatg gtaacaacgt tgcgcaaact attaactggc 7021 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt 7081 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 7141 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc 7201 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag 7261 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca 7321 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 7381 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 7441 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 7501 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 7561 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt 7621 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 7681 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 7741 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 7801 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 7861 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 7921 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 7981 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 8041 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 8101 tgcggttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 8161 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca 8221 gtgagcgagg aagcggaag //