LOCUS Exported 9258 bp ds-DNA circular SYN 30-JUL-2019 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9258) AUTHORS . TITLE Direct Submission JOURNAL Exported Wednesday, Jul 31, 2019 from SnapGene Viewer 4.3.8 https://www.snapgene.com FEATURES Location/Qualifiers source 1..9258 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 62..302 /label=U6 promoter primer_bind 107..131 /label=pORANGE sequencing primer FW misc_feature 311..331 /label=Target Sequence misc_RNA 332..407 /label=gRNA scaffold misc_feature 408..418 /label=Termination sequence misc_feature 503..522 /label=Target Sequence misc_feature 523..525 /label=PAM CDS 526..534 /codon_start=1 /label=Linker /translation="SGG" CDS 535..1248 /codon_start=1 /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" CDS 1249..1254 /codon_start=1 /label=Linker /translation="GS" misc_feature 1256..1275 /label=Target sequence misc_feature 1276..1278 /label=PAM enhancer 1323..1608 /label=CMV enhancer primer_bind complement(1331..1350) /label=pORANGE sequencing primer RV promoter 1610..1887 /label=chicken beta-actin promoter intron 1888..2115 /label=hybrid intron misc_feature 2133..2135 /label=START CDS 2136..2162 /codon_start=1 /label=HA /translation="YPYDVPDYA" CDS 2166..2186 /codon_start=1 /label=SV40 NLS /translation="PKKKRKV" CDS 2196..6296 /codon_start=1 /label=Cas9 /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 6300..6320 /codon_start=1 /label=SV40 NLS /translation="PKKKRKV" polyA_signal 6366..6573 /label=bGH poly(A) signal repeat_region 6582..6722 /label=AAV2 ITR rep_origin 6797..7252 /direction=RIGHT /label=f1 ori /note="f1 stays for filamentous phages. It initiates a ""rolling circle"" replication in phage-infected bacteria and is useful if there is a need to produce vector as a single-stranded DNA" promoter 7534..7638 /label=AmpR promoter CDS 7639..8499 /codon_start=1 /label=AmpR /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" rep_origin 8670..9258 /direction=RIGHT /label=ori /note="Ori for replication in E.coli" ORIGIN 1 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 61 tgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga 121 gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag 181 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca 241 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg 301 acgaaacacc gaatcagagt ctctctcggg cgttttagag ctagaaatag caagttaaaa 361 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttgttttag 421 agctagaaat agcaagttaa aataaggcta gtccgttttt agcgcgtgcg ccaattctgc 481 agacaaatgg ctctagaagc ttaatcagag tctctctcgg gcgggagcgg tggagtgagc 541 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta 601 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg 661 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc 721 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac 781 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac 841 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc 901 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag 961 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag 1021 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac 1081 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc 1141 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag 1201 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaaggg ttcaaaatca 1261 gagtctctct cgggcgggct cgaggatcct aggacgcgtc ctaggacgcg tccagaggta 1321 cccgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca 1381 ttgacgtcaa tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta 1441 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt 1501 gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc agtacatgac cttatgggac 1561 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc 1621 cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat tttgtattta 1681 tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggg gcgcgcgcca 1741 ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc 1801 aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc 1861 tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc 1921 cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag 1981 gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agctgagcaa gaggtaaggg 2041 tttaagggat ggttggttgg tggggtatta atgtttaatt acctggagca cctgcctgaa 2101 atcacttttt ttcaggttgg accggtgcca ccatgtaccc atacgatgtt ccagattacg 2161 cttcgccgaa gaaaaagcgc aaggtcgaag cgtccgacaa gaagtacagc atcggcctgg 2221 acatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca 2281 agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag 2341 ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa 2401 gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga 2461 tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg 2521 ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg 2581 agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg 2641 acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga 2701 tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc 2761 agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg 2821 ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc 2881 ccggcgagaa gaagaatggc ctgttcggca acctgattgc cctgagcctg ggcctgaccc 2941 ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca 3001 cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt 3061 ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca 3121 ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc 3181 aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga 3241 ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg 3301 aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc 3361 tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca 3421 tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt 3481 acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct 3541 actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg 3601 aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct tccgcccaga 3661 gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca 3721 agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg 3781 tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg 3841 acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca 3901 agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct 3961 ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc ctggacaatg 4021 aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt gaggacagag 4081 agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc 4141 agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg atcaacggca 4201 tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac ggcttcgcca 4261 acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag gacatccaga 4321 aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat ctggccggca 4381 gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag 4441 tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag aaccagacca 4501 cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag 4561 agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg cagaacgaga 4621 agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag gaactggaca 4681 tcaaccggct gtccgactac gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg 4741 actccatcga caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg 4801 tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg ctgaacgcca 4861 agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg 4921 aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag atcacaaagc 4981 acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat gacaagctga 5041 tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc cggaaggatt 5101 tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac gcctacctga 5161 acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt 5221 acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag caggaaatcg 5281 gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc aagaccgaga 5341 ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac ggcgaaaccg 5401 gggagatcgt gtgggataag ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc 5461 cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc aaagagtcta 5521 tcctgcccaa gaggaacagc gataagctga tcgccagaaa gaaggactgg gaccctaaga 5581 agtacggcgg cttcgacagc cccaccgtgg cctattctgt gctggtggtg gccaaagtgg 5641 aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc accatcatgg 5701 aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc tacaaagaag 5761 tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg gaaaacggcc 5821 ggaagagaat gctggcctct gccggcgaac tgcagaaggg aaacgaactg gccctgccct 5881 ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag ggctcccccg 5941 aggataatga gcagaaacag ctgtttgtgg aacagcacaa gcactacctg gacgagatca 6001 tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat ctggacaaag 6061 tgctgtccgc ctacaacaag caccgggata agcccatcag agagcaggcc gagaatatca 6121 tccacctgtt taccctgacc aatctgggag cccctgccgc cttcaagtac tttgacacca 6181 ccatcgaccg gaagaggtac accagcacca aagaggtgct ggacgccacc ctgatccacc 6241 agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga ggcgacagcc 6301 ccaagaagaa gagaaaggtg gaggccagct aagaattcta actagagctc gctgatcagc 6361 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt 6421 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca 6481 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga 6541 ggattgggaa gagaatagca ggcatgctgg ggagcggccg caggaacccc tagtgatgga 6601 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc 6661 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca 6721 ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg 6781 tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt 6841 acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc 6901 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct 6961 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat 7021 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc 7081 acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc 7141 tattcttttg atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg 7201 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc 7261 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca 7321 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg 7381 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga 7441 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct 7501 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 7561 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 7621 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt 7681 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct 7741 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc 7801 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 7861 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac 7921 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 7981 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 8041 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 8101 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac 8161 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc 8221 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt 8281 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 8341 gccggtgagc gtggaagccg cggtatcatt gcagcactgg ggccagatgg taagccctcc 8401 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag 8461 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca 8521 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 8581 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 8641 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 8701 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 8761 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt 8821 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 8881 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 8941 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 9001 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 9061 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 9121 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 9181 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 9241 gggcggagcc tatggaaa //