LOCUS Exported 8243 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to 8243) TITLE New frontiers to cure Alport syndrome: COL4A3 and COL4A5 gene editing in podocyte-lineage cells JOURNAL Unpublished REFERENCE 2 (bases 1 to 8243) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8243 /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 259..388 /label=AAV2 ITR promoter 621..824 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 775..795 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" CDS 889..954 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 961..981 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1006..5106 /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 5107..5154 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5250..5267) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5256..5463 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5551..5680 /label=AAV2 ITR primer_bind complement(5699..5716) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(5699..5715) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5708..5730) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 5857..6312 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5944..5963) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6154..6175 /label=F1ori-F /note="F1 origin, forward primer" promoter 6338..6442 /gene="bla" /label=AmpR promoter CDS 6443..7303 /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(6661..6680) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7474..8062 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7963..7982 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 8216..8233 /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 attaaggcct taattaggct gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc 301 gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga 361 gtggccaact ccatcactag gggttccttg tagttaatga ttaacccgcc atgctactta 421 tctacgtagc catgctctag gaagatcgga attcgccctt aagctagtcg acggtatcga 481 taagcttgat tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 541 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 601 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 661 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 721 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 781 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc 841 agatccgcta gagcttggat cccctggagg gccgaaatca gggggatgga ctataaggac 901 cacgacggag actacaagga tcatgatatt gattacaaag acgatgacga taagatggcc 961 ccaaagaaga agcggaaggt cggtatccac ggagtcccag cagccgacaa gaagtacagc 1021 atcggcctgg acatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag 1081 gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac 1141 ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga 1201 accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc 1261 agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg 1321 gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg 1381 gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc 1441 gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc 1501 cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc 1561 cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg 1621 gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc 1681 gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc cctgagcctg 1741 ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg 1801 agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac 1861 gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg 1921 agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac 1981 gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag 2041 tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga 2101 gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc 2161 gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac 2221 aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag 2281 gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc 2341 cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc 2401 agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct 2461 tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag 2521 gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa 2581 gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag 2641 gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag 2701 gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg 2761 ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc 2821 ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt 2881 gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa 2941 gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg 3001 atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac 3061 ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag 3121 gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat 3181 ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag 3241 ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag 3301 aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag 3361 ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg 3421 cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag 3481 gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc tcagagcttt 3541 ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag 3601 agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg 3661 ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc 3721 ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag 3781 atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat 3841 gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc 3901 cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac 3961 gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc 4021 gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag 4081 caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc 4141 aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac 4201 ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt gcggaaagtg 4261 ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc 4321 aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa gaaggactgg 4381 gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt gctggtggtg 4441 gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc 4501 accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc 4561 tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg 4621 gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg aaacgaactg 4681 gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag 4741 ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa gcactacctg 4801 gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat 4861 ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag agagcaggcc 4921 gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc cttcaagtac 4981 tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct ggacgccacc 5041 ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga 5101 ggcgacaaaa ggccggcggc cacgaaaaag gccggccagg caaaaaagaa aaagtaagaa 5161 ttcctagagc tcgctgaccc cctgatttcg gccctccagg ccgcggcctg ctgccggctc 5221 tgcggcctct tccgcgtctt cgagatctgc ctcgactgtg ccttctagtt gccagccatc 5281 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct 5341 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg 5401 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg 5461 ggactcgagt taagggcgaa ttcccgataa ggatcttcct agagcatggc tacgtagata 5521 agtagcatgg cgggttaatc attaactaca aggaacccct agtgatggag ttggccactc 5581 cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg 5641 gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ccttaattaa cctaattcac 5701 tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc 5761 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc 5821 cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa 5881 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc 5941 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag 6001 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca 6061 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc 6121 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa 6181 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct 6241 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa 6301 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 6361 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 6421 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 6481 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga 6541 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa 6601 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct 6661 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat 6721 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga 6781 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc 6841 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat 6901 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa 6961 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac 7021 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa 7081 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc 7141 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc 7201 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag 7261 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta 7321 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa 7381 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc 7441 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat 7501 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga 7561 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt 7621 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata 7681 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac 7741 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg 7801 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg 7861 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag 7921 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct 7981 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc 8041 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt 8101 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg 8161 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga 8221 gtcagtgagc gaggaagcgg aag //