LOCUS Exported 9541 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expression of dead/inactive increased fidelity eSpCas9 (1.1)-VP64-6xHis in bacterial cells. ACCESSION . VERSION . KEYWORDS pET-deSpCas9-VP64-6xHis SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9541) AUTHORS Kulcsar PI, Talas A, Huszar K, Ligeti Z, Toth E, Weinhardt N, Fodor E, Welker E TITLE Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage. JOURNAL Genome Biol. 2017 Oct 6;18(1):190. doi: 10.1186/s13059-017-1318-8. PUBMED 28985763 REFERENCE 2 (bases 1 to 9541) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9541 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 1..20 /label=T7 /note="T7 promoter, forward primer" promoter 1..19 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 20..44 /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)." RBS 59..81 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 4198..4218 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4225..4290 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 4321..4470 /codon_start=1 /product="tetrameric repeat of the minimal activation domain of herpes simplex virus VP16 (Beerli et al., 1998)" /label=VP64 /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF DLDML" CDS 4471..4488 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 4507..4551 /codon_start=1 /product="affinity and epitope tag derived from pancreatic ribonuclease A" /label=S-Tag /translation="KETAAAKFERQHMDS" primer_bind complement(4589..4607) /label=T7 Term /note="T7 terminator, reverse primer" terminator 4603..4650 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 4687..5142 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4774..4793) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4984..5005 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(5257..6117) /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 5880..5899 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6118..6210) /gene="bla" /label=AmpR promoter rep_origin 6291..6879 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6780..6799 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 7033..7050 /label=L4440 /note="L4440 vector, forward primer" misc_feature 7065..7204 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(7130..7149) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7290..7312 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(7306..7497) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(8069..9151) /codon_start=1 /gene="lacI" /product="lac repressor" /label=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)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" primer_bind 9113..9132 /label=LacI-R /note="LacI, reverse primer" promoter complement(9152..9229) /gene="lacI" /label=lacI promoter primer_bind 9435..9454 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" ORIGIN 1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt 61 tgtttaactt taagaaggag atataccatg gataagaaat actcaatagg cttagctatc 121 ggcacaaata gcgtcggatg ggcggtgatc actgatgaat ataaggttcc gtctaaaaag 181 ttcaaggttc tgggaaatac agaccgccac agtatcaaaa aaaatcttat aggggctctt 241 ttatttgaca gtggagagac agcggaagcg actcgtctca aacggacagc tcgtagaagg 301 tatacacgtc ggaagaatcg tatttgttat ctacaggaga ttttttcaaa tgagatggcg 361 aaagtagatg atagtttctt tcatcgactt gaagagtctt ttttggtgga agaagacaag 421 aagcatgaac gtcatcctat ttttggaaat atagtagatg aagttgctta tcatgagaaa 481 tatccaacta tctatcatct gcgaaaaaaa ttggtagatt ctactgataa agcggatttg 541 cgcttaatct atttggcctt agcgcatatg attaagtttc gtggtcattt tttgattgag 601 ggagatttaa atcctgataa tagtgatgtg gacaaactat ttatccagtt ggtacaaacc 661 tacaatcaat tatttgaaga aaaccctatt aacgcaagtg gagtagatgc taaagcgatt 721 ctttctgcac gattgagtaa atcaagacga ttagaaaatc tcattgctca gctccccggt 781 gagaagaaaa atggcttatt tgggaatctc attgctttgt cattgggttt gacccctaat 841 tttaaatcaa attttgattt ggcagaagat gctaaattac agctttcaaa agatacttac 901 gatgatgatt tagataattt attggcgcaa attggagatc aatatgctga tttgtttttg 961 gcagctaaga atttatcaga tgctatttta ctttcagata tcctaagagt aaatactgaa 1021 ataactaagg ctcccctatc agcttcaatg attaaacgct acgatgaaca tcatcaagac 1081 ttgactcttt taaaagcttt agttcgacaa caacttccag aaaagtataa agaaatcttt 1141 tttgatcaat caaaaaacgg atatgcaggt tatattgatg ggggagctag ccaggaagag 1201 ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg 1261 aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg cagcatcccc 1321 caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga tttttaccca 1381 ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat cccctactac 1441 gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa 1501 accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc 1561 atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac 1621 agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa atacgtgacc 1681 gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat cgtggacctg 1741 ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa 1801 atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg 1861 ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa 1921 aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg 1981 atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg 2041 aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgg 2101 gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga 2161 aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat ccagaaagcc 2221 caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc 2281 gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg 2341 ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag 2401 aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat caaagagctg 2461 ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg 2521 tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact ggacatcaac 2581 cggctgtccg actacgatgt ggacgccatc gtgcctcaga gctttctggc cgacgactcc 2641 atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga caacgtgccc 2701 tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa cgccaagctg 2761 attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct gagcgaactg 2821 gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac aaagcacgtg 2881 gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa gctgatccgg 2941 gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag 3001 ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta cctgaacgcc 3061 gtcgtgggaa ccgccctgat caaaaagtac cctgcgctgg aaagcgagtt cgtgtacggc 3121 gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga aatcggcaag 3181 gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac cgagattacc 3241 ctggccaacg gcgagatccg gaaggcgcct ctgatcgaga caaacggcga aaccggggag 3301 atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag catgccccaa 3361 gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg 3421 cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggatcc aaaaaaatat 3481 ggtggttttg atagtccaac ggtagcttat tcagtcctag tggttgctaa ggtggaaaaa 3541 gggaaatcga agaagttaaa atccgttaaa gagttactag ggatcacaat tatggaaaga 3601 agttcctttg aaaaaaatcc gattgacttt ttagaagcta aaggatataa ggaagttaaa 3661 aaagacttaa tcattaaact acctaaatat agtctttttg agttagaaaa cggtcgtaaa 3721 cggatgctgg ctagtgccgg agaattacaa aaaggaaatg agctggctct gccaagcaaa 3781 tatgtgaatt ttttatattt agctagtcat tatgaaaagt tgaagggtag tccagaagat 3841 aacgaacaaa aacaattgtt tgtggagcag cataagcatt atttagatga gattattgag 3901 caaatcagtg aattttctaa gcgtgttatt ttagcagatg ccaatttaga taaagttctt 3961 agtgcatata acaaacatag agacaaacca atacgtgaac aagcagaaaa tattattcat 4021 ttatttacgt tgacgaatct tggagctccc gctgctttta aatattttga tacaacaatt 4081 gatcgtaaac gatatacgtc tacaaaagaa gttttagatg ccactcttat ccatcaatcc 4141 atcactggtc tttatgaaac acgcattgat ttgagtcagc taggaggtga cggttctccc 4201 aagaagaaga ggaaagtctc gagcgactac aaagaccatg acggtgatta taaagatcat 4261 gacatcgatt acaaggatga cgatgacaag gctgcaggag gcggtggaag cgggcgcgcc 4321 gacgcgctgg acgatttcga tctcgacatg ctgggttctg atgccctcga tgactttgac 4381 ctggatatgt tgggaagcga cgcattggat gactttgatc tggacatgct cggctccgat 4441 gctctggacg atttcgatct cgatatgtta catcaccacc accatcacta atgactcgag 4501 tctggtaaag aaaccgctgc tgcgaaattt gaacgccagc acatggactc gtctactagc 4561 gcagcttaat taacctaggc tgctgccacc gctgagcaat aactagcata accccttggg 4621 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg 4681 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg 4741 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc 4801 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc 4861 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta 4921 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta 4981 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg 5041 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa 5101 aatttaacgc gaattttaac aaaatattaa cgtttacaat ttctggcggc acgatggcat 5161 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 5221 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc 5281 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta 5341 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga 5401 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg 5461 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc 5521 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat 5581 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag 5641 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat 5701 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa 5761 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa 5821 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga 5881 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg 5941 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc 6001 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 6061 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact 6121 cttccttttt caatcatgat tgaagcattt atcagggtta ttgtctcatg agcggataca 6181 tatttgaatg tatttagaaa aataaacaaa taggtcatga ccaaaatccc ttaacgtgag 6241 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct 6301 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 6361 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg 6421 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct 6481 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc 6541 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg 6601 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 6661 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg 6721 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg 6781 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga 6841 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt 6901 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct 6961 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga 7021 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt 7081 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct 7141 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc 7201 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc 7261 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc 7321 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga 7381 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa 7441 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga 7501 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag 7561 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg 7621 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca 7681 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca 7741 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg 7801 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct 7861 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc 7921 tagccgggtc ctcaacgaca ggagcacgat catgctagtc atgccccgcg cccaccggaa 7981 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt 8041 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc 8101 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg 8161 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct 8221 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct 8281 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca 8341 ctaccgagat gtccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca 8401 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt 8461 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct 8521 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag 8581 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca 8641 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag 8701 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct 8761 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca 8821 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac 8881 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca 8941 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc 9001 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag 9061 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact 9121 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg 9181 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga 9241 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg 9301 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc 9361 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc 9421 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg 9481 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tcgatctcga tcccgcgaaa 9541 t //