LOCUS 40924_435 10437 bp DNA circular SYN 17-DEC-2018 DEFINITION Cloning vector CasYFP, complete sequence. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10437) AUTHORS Hahn F, Mantegazza O, Greiner A, Hegemann P, Eisenhut M, Weber AP. TITLE An Efficient Visual Screen for CRISPR/Cas9 Activity in Arabidopsis thaliana JOURNAL Front Plant Sci 8, 39 (2017) PUBMED 28174584 REFERENCE 2 (bases 1 to 10437) AUTHORS Hahn F, Mantegazza O, Eisenhut M, Greiner A, Hegemann P, Weber APM. TITLE Direct Submission JOURNAL Submitted (03-NOV-2016) Plant Biochemistry, Heinrich Heine University Dusseldorf, Universitatsstr. 1, Dusseldorf 40225, Germany REFERENCE 3 (bases 1 to 10437) TITLE Direct Submission REFERENCE 4 (bases 1 to 10437) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Front Plant Sci 8, 39 (2017)" COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted (03-NOV-2016) Plant Biochemistry, Heinrich Heine University Dusseldorf, Universitatsstr. 1, Dusseldorf 40225, Germany" COMMENT SGRef: number: 3; type: "Journal Article" FEATURES Location/Qualifiers source 1..10437 /mol_type="other DNA" /organism="synthetic DNA construct" rep_origin 4..362 /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(385..401) /label=SK primer /note="common sequencing primer, one of multiple similar variants" misc_RNA complement(785..860) /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" regulatory complement(881..1380) /label=U6/C8 /note="U6/C8" /regulatory_class="promoter" regulatory 1441..2254 /label=from Chlamydomonas reinhardtii /note="from Chlamydomonas reinhardtii" /regulatory_class="promoter" CDS 2273..2293 /label=SV40 NLS /note="nuclear localization signal of SV40 (simian virus 40) large T antigen" CDS 2291..6394 /label=Cas9 /note="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" CDS 6401..7114 /label=EYFP /note="enhanced YFP" CDS 7130..7186 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 7187..7987 /codon_start=1 /product="aminoglycoside 3'-phosphotransferase VIII" /function="Selection marker in Chlamydomonas reinhardtii" /label=aminoglycoside 3'-phosphotransferase VIII /note="selection marker aminoglycoside 3'-phosphotransferase gene (aph) from Streptomyces rimosus" /protein_id="AQN67803.1" /translation="DDALRALRGRYPGCEWVVVEDGASGAGVYRLRGGGRELFVKVAAL GAGVGLLGEAERLVWLAEVGIPVPRVVEGGGDERVAWLVTEAVPGRPASARWPREQRLD VAVALAGLARSLHALDWERCPFDRSLAVTVPQAARAVAEGSVDLEDLDEERKGWSGERL LAELERTRPADEDLAVCHGDLCPDNVLLDPRTCEVTGLIDVGRVGRADRHSDLALVLRE LAHEEDPWFGPECSAAFLREYGRGWDGAVSEEKLAFYRLLDEFF" regulatory 7991..8230 /regulatory_class="terminator" promoter complement(8249..8267) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(8288..8304) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 8312..8328 /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)." promoter complement(8336..8366) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind complement(8381..8402) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(8690..9278) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(9452..10309) /label=AmpR /note="beta-lactamase" promoter complement(10310..10414) /label=AmpR promoter ORIGIN 1 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 61 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg 121 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat 181 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg 241 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata 301 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt 361 taatcgaatt cctgcagccc gggggatcca ctagttctag aaagatggaa ggggcaggac 421 acccacgccg cagcaccgcc tgcaccagca tcccttgcgc aggtcgtgat atagcagcaa 481 gtaagtgtaa atccaggttc cggcgaagac acgcacccag gtagaccctg gtcatgtggt 541 cactgagctt ccctgagcct ctcgcgctca ctgagcccct ccccctcact cgcgctcagt 601 tgcccgccca ccgtgaacac gcgcgagcca cgcgacaatg gactcgtcgt cacgcacatg 661 ttgcaattgc cgtgttatgc cgcaccgcag tacccagggg acgcgggaag atggtgcggg 721 gagagggcca gggcggtcgg ccagggtcga actgggggtc tgcgcgccga gacgaaaaaa 781 atttgcaccg actcggtgcc actttttcaa gttgataacg gactagcctt attttaactt 841 gctatttcta gctctaaaac ccacgaagtc ccgggagaac gacagtgctc gtcggtgtaa 901 tatacatatc actagggcgc aaaagtcgcg tccaaacaga caccggcgcc agcccgacct 961 ttcccgcgcg ggagcgcgtt gatcccgacc cggtaagtcc catcgtggaa cagtagcccc 1021 aaggcccctt ccatagcgca gatgttcctg ggcgctctcc agcgtgcatg cgggttctag 1081 gtagccacgc ccacgctgcg aggctgggaa aaaggcccat tgtgacagca agccagcttt 1141 cgcgccattg gttgccggcg acagcccagt ggtgtggcag cttgggtatg catagcttgt 1201 tggggtacgg gtgggcaatg actcccaacc ttgcgcgcgt aggatttatg agggcatcct 1261 gctccctgcg caaaagcatg aatttgcacc catgcaatgc gcgaggggag cagctaccca 1321 ccgcgcgctg ccctgctgcc aggtatcaac tggattatca atcccaatac ccaggcattt 1381 atcttgctga aaaactcgag ccatccggaa gatctggcgg ccgccaccgc ggtggagctc 1441 cacacacctg cccgtctgcc tgacaggaag tgaacgcatg tcgagggagg cctcaccmat 1501 cgtcacacga gccctcgtca gaaacacgtc tccgccacgc tctccctctc acggccgacc 1561 ccgcagccct tttgcccttt cctaggccac cgacaggacc caggcgctct cagcatgcct 1621 caacaacccg tactcgtgcc agcggtgccc ttgtgctggt gatcgcttgg aagcgcatgc 1681 gaagacgaag gggcggagca ggcggcctgg ctgttcgaag ggctcgccgc cagttcgggt 1741 gcctttctcc acgcgcgcct ccacacctac cgatgcgtga aggcaggcaa atgctcatgt 1801 ttgcccgaac tcggagtcct taaaaagccg cttcttgtcg tcgttccgag acatgttagc 1861 agatcgcagt gccacctttc ctgacgcgct cggccccata ttcggacgca attgtcattt 1921 gtagcacaat tggagcaaat ctggcgaggc agtaggcttt taagttgcaa ggcgagagag 1981 caaagtggga cgcggcgtga ttattggtat ttacgcgacg gcccggcgcg ttagcggccc 2041 ttcccccagg ccagggacga ttatgtatca atattgttgc gttcgggcac tcgtgcgagg 2101 gctcctgcgg gctggggagg gggatctggg aattggaggt acgaccgaga tggcttgctc 2161 ggggggaggt ttcctcgccg agcaagccag ggttaggtgt tgcgctcttg actcgttgtg 2221 cattctagga ccccactgct actcacaaca agcccatatg gccagcagcc cccccaagaa 2281 gaagcgcaag gtcgacaaga agtactccat cggcctggac atcggcacga acagcgtggg 2341 ctgggccgtg atcaccgacg agtacaaggt gccgtccaag aagttcaagg tgctgggcaa 2401 caccgaccgc cacagcatca agaagaacct gatcggcgcc ctgctgttcg actcgggcga 2461 gaccgccgag gcgacgcgcc tgaagcgcac cgcgcgccgc cgctacacgc gccgcaagaa 2521 ccgcatctgc tacctgcagg agatcttcag caacgagatg gccaaggtgg acgactcgtt 2581 cttccaccgc ctggaggagt ccttcctggt ggaggaggac aagaagcacg agcgccaccc 2641 catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccga cgatctacca 2701 cctgcgcaag aagctggtgg acagcaccga caaggcggac ctgcgcctga tctacctggc 2761 cctggcgcac atgatcaagt tccgcggcca cttcctgatc gagggcgacc tgaaccccga 2821 caactcggac gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga 2881 ggagaacccg atcaacgcct cgggcgtgga cgccaaggcg atcctgtccg cgcgcctgtc 2941 caagagccgc cgcctggaga acctgatcgc ccagctgccc ggcgagaaga agaacggcct 3001 gttcggcaac ctgatcgcgc tgagcctggg cctgacgccg aacttcaagt cgaacttcga 3061 cctggccgag gacgcgaagc tgcagctgag caaggacacc tacgacgacg acctggacaa 3121 cctgctggcc cagatcggcg accagtacgc ggacctgttc ctggccgcga agaacctgtc 3181 ggacgccatc ctgctgtccg acatcctgcg cgtgaacacc gagatcacga aggcccccct 3241 gtcggcgtcc atgatcaagc gctacgacga gcaccaccag gacctgaccc tgctgaaggc 3301 gctggtgcgc cagcagctgc cggagaagta caaggagatc ttcttcgacc agtccaagaa 3361 cggctacgcc ggctacatcg acggcggcgc cagccaggag gagttctaca agttcatcaa 3421 gcccatcctg gagaagatgg acggcacgga ggagctgctg gtgaagctga accgcgagga 3481 cctgctgcgc aagcagcgca ccttcgacaa cggcagcatc ccccaccaga tccacctggg 3541 cgagctgcac gccatcctgc gccgccagga ggacttctac ccgttcctga aggacaaccg 3601 cgagaagatc gagaagatcc tgacgttccg catcccctac tacgtgggcc cgctggctcg 3661 cggcaactcc cgcttcgcgt ggatgacccg caagagcgag gagaccatca cgccctggaa 3721 cttcgaggag gtggtggaca agggcgccag cgcgcagtcg ttcatcgagc gcatgaccaa 3781 cttcgacaag aacctgccca acgagaaggt gctgccgaag cactccctgc tgtacgagta 3841 cttcaccgtg tacaacgagc tgacgaaggt gaagtacgtg accgagggca tgcgcaagcc 3901 cgccttcctg agcggcgagc agaagaaggc gatcgtggac ctgctgttca agaccaaccg 3961 caaggtgacg gtgaagcagc tgaaggagga ctacttcaag aagatcgagt gcttcgacag 4021 cgtggagatc tcgggcgtgg aggaccgctt caacgccagc ctgggcacct accacgacct 4081 gctgaagatc atcaaggaca aggacttcct ggacaacgag gagaacgagg acatcctgga 4141 ggacatcgtg ctgaccctga cgctgttcga ggaccgcgag atgatcgagg agcgcctgaa 4201 gacgtacgcc cacctgttcg acgacaaggt gatgaagcag ctgaagcgcc gccgctacac 4261 cggctggggc cgcctgagcc gcaagctgat caacggcatc cgcgacaagc agtcgggcaa 4321 gaccatcctg gacttcctga agtccgacgg cttcgcgaac cgcaacttca tgcagctgat 4381 ccacgacgac tcgctgacct tcaaggagga catccagaag gcccaggtgt cgggccaggg 4441 cgactccctg cacgagcaca tcgccaacct ggcgggctcc cccgcgatca agaagggcat 4501 cctgcagacc gtgaaggtgg tggacgagct ggtgaaggtg atgggccgcc acaagccgga 4561 gaacatcgtg atcgagatgg cccgcgagaa ccagaccacg cagaagggcc agaagaacag 4621 ccgcgagcgc atgaagcgca tcgaggaggg catcaaggag ctgggctcgc agatcctgaa 4681 ggagcacccc gtggagaaca cccagctgca gaacgagaag ctgtacctgt actacctgca 4741 gaacggccgc gacatgtacg tggaccagga gctggacatc aaccgcctgt ccgactacga 4801 cgtggaccac atcgtgcccc agagcttcct gaaggacgac tcgatcgaca acaaggtgct 4861 gacccgcagc gacaagaacc gcggcaagag cgacaacgtg ccgtcggagg aggtggtgaa 4921 gaagatgaag aactactggc gccagctgct gaacgccaag ctgatcacgc agcgcaagtt 4981 cgacaacctg accaaggccg agcgcggcgg cctgtcggag ctggacaagg cgggcttcat 5041 caagcgccag ctggtggaga cccgccagat cacgaagcac gtggcgcaga tcctggactc 5101 ccgcatgaac acgaagtacg acgagaacga caagctgatc cgcgaggtga aggtgatcac 5161 cctgaagtcc aagctggtca gcgacttccg caaggacttc cagttctaca aggtgcgcga 5221 gatcaacaac taccaccacg cccacgacgc gtacctgaac gccgtggtgg gcaccgcgct 5281 gatcaagaag taccccaagc tggagagcga gttcgtgtac ggcgactaca aggtgtacga 5341 cgtgcgcaag atgatcgcca agtcggagca ggagatcggc aaggccaccg cgaagtactt 5401 cttctactcc aacatcatga acttcttcaa gaccgagatc acgctggcca acggcgagat 5461 ccgcaagcgc cccctgatcg agaccaacgg cgagacgggc gagatcgtgt gggacaaggg 5521 ccgcgacttc gcgaccgtgc gcaaggtgct gagcatgccc caggtgaaca tcgtgaagaa 5581 gaccgaggtg cagacgggcg gcttctccaa ggagagcatc ctgccgaagc gcaactcgga 5641 caagctgatc gcccgcaaga aggactggga ccccaagaag tacggcggct tcgactcccc 5701 gaccgtggcc tacagcgtgc tggtggtggc gaaggtggag aagggcaagt ccaagaagct 5761 gaagagcgtg aaggagctgc tgggcatcac catcatggag cgcagctcgt tcgagaagaa 5821 ccccatcgac ttcctggagg cgaagggcta caaggaggtg aagaaggacc tgatcatcaa 5881 gctgccgaag tactcgctgt tcgagctgga gaacggccgc aagcgcatgc tggcctccgc 5941 gggcgagctg cagaagggca acgagctggc cctgcccagc aagtacgtga acttcctgta 6001 cctggcgtcc cactacgaga agctgaaggg ctcgccggag gacaacgagc agaagcagct 6061 gttcgtggag cagcacaagc actacctgga cgagatcatc gagcagatct cggagttctc 6121 caagcgcgtg atcctggccg acgcgaacct ggacaaggtg ctgagcgcct acaacaagca 6181 ccgcgacaag cccatccgcg agcaggcgga gaacatcatc cacctgttca ccctgacgaa 6241 cctgggcgcc ccggccgcgt tcaagtactt cgacaccacg atcgaccgca agcgctacac 6301 cagcacgaag gaggtgctgg acgcgaccct gatccaccag agcatcaccg gcctgtacga 6361 gacgcgcatc gacctgtcgc agctgggcgg cgacaccggt gtgagcaagg gcgaggagct 6421 gttcacgggc gtggtgccca tcctggtgga gctggacggc gacgtgaacg gccacaagtt 6481 cagcgtgtcc ggcgagggcg agggcgacgc cacctacggc aagctgaccc tgaagttcat 6541 ctgcacgacc ggcaagctgc ccgtgccctg gcccaccctg gtgaccacct tcggctacgg 6601 cctgcagtgc ttcgcccgct accccgacca catgaagcag cacgacttct tcaagagcgc 6661 catgccggag ggctacgtgc aggagcgcac catcttcttc aaggacgacg gcaactacaa 6721 gacccgcgcg gaggtgaagt tcgagggcga cacgctggtg aaccgcatcg agctgaaggg 6781 catcgacttc aaggaggacg gcaacatcct gggccacaag ctggagtaca actacaacag 6841 ccacaacgtg tacatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat 6901 ccgccacaac atcgaggacg gctcggtgca gctggcggac cactaccagc agaacacccc 6961 catcggcgac ggcccggtgc tgctgcccga caaccactac ctgtcctacc agagcgccct 7021 gtccaaggac cccaacgaga agcgcgacca catggtgctg ctggagttcg tgaccgccgc 7081 cggcatcacc ctgggcatgg acgagctgta caaggaattc ggcagcggcg ccaccaactt 7141 cagcctgctg aagcaggccg gcgacgtgga ggagaacccc ggccccgacg atgcgttgcg 7201 tgcactgcgg ggtcggtatc ccggttgtga gtgggttgtt gtggaggatg gggcctcggg 7261 ggctggtgtt tatcggcttc ggggtggtgg gcgggagttg tttgtcaagg tggcagctct 7321 gggggccggg gtgggcttgt tgggtgaggc tgagcggctg gtgtggttgg cggaggtggg 7381 gattcccgta cctcgtgttg tggagggtgg tggggacgag agggtcgcct ggttggtcac 7441 cgaagcggtt ccggggcgtc cggccagtgc gcggtggccg cgggagcagc ggctggacgt 7501 ggcggtggcg ctcgcggggc tcgctcgttc gctgcacgcg ctggactggg agcggtgtcc 7561 gttcgatcgc agtctcgcgg tgacggtgcc gcaggcggcc cgtgctgtcg ctgaagggag 7621 cgtcgacttg gaggatctgg acgaggagcg gaaggggtgg tcgggggagc ggcttctcgc 7681 cgagctggag cggactcggc ctgcggacga ggatctggcg gtttgccacg gtgacctgtg 7741 cccggacaac gtgctgctcg accctcgtac ctgcgaggtg accgggctga tcgacgtggg 7801 gcgggtcggc cgtgcggacc ggcactccga tctcgcgctg gtgctgcgcg agctggccca 7861 cgaggaggac ccgtggttcg ggccggagtg ttccgcggcg ttcctgcggg agtacgggcg 7921 cgggtgggat ggggcggtat cggaggaaaa gctggcgttt taccggctgt tggacgagtt 7981 cttctgataa ggatccccgc tccgtgtaaa tggaggcgct cgttgatctg agccttgccc 8041 cctgacgaac ggcggtggat ggaagatact gctctcaagt gctgaagcgg tagcttagct 8101 ccccgtttcg tgctgatcag tctttttcaa cacgtaaaaa gcggaggagt tttgcaattt 8161 tgttggttgt aacgatcctc cgttgatttt ggcctctttc tccatgggcg ggctgggcgt 8221 atttgaagcg ggtacccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt 8281 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca 8341 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat 8401 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt 8461 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct 8521 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 8581 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 8641 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 8701 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 8761 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 8821 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 8881 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 8941 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 9001 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 9061 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 9121 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 9181 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 9241 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 9301 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 9361 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 9421 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 9481 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 9541 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 9601 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 9661 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 9721 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 9781 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 9841 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 9901 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 9961 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 10021 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 10081 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 10141 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 10201 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 10261 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 10321 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 10381 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccac //