LOCUS Exported 8468 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION CRISPR plasmid for expression of Cas9 and gRNA targeting human transcription factor ESR1. ACCESSION . VERSION . KEYWORDS pESR1.1.0-gDNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8468) TITLE White lab ENCODE plasmids to tag human transcription factors with GFP REFERENCE 2 (bases 1 to 8468) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8468 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 169..454 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" intron 694..921 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 933..942 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 942..1007 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1014..1034 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1059..5159 /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 5160..5207 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5235..5252) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5241..5448 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5457..5597 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5457..5586 /label=AAV2 ITR rep_origin 5672..6127 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5759..5778) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5969..5990 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6144..6163) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6263..6285 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6323..6341) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6409..6513 /gene="bla" /label=AmpR promoter CDS 6514..7374 /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(6732..6751) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7545..8133 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8034..8053 /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 8195..8435 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 8195..8215 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 8366..8385 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 8465..72 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" ORIGIN 1 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca 61 ccgagtcggt gcttttttgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg 121 tttttagcgc gtgcgccaat tctgcagaca aatggctcta gaggtacccg ttacataact 181 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaatagt 241 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca 301 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg 361 taaatggccc gcctggcatt gtgcccagta catgacctta tgggactttc ctacttggca 421 gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt 481 cactctcccc atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt 541 attttgtgca gcgatggggg cggggggggg gggggcgggg cgaggcggag aggtgcggcg 601 gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg 661 cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc 721 gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc 781 ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg 841 taagggttta agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg 901 cctgaaatca ctttttttca ggttggaccg gtgccaccat ggactataag gaccacgacg 961 gagactacaa ggatcatgat attgattaca aagacgatga cgataagatg gccccaaaga 1021 agaagcggaa ggtcggtatc cacggagtcc cagcagccga caagaagtac agcatcggcc 1081 tggacatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca 1141 gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg 1201 gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca 1261 gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg 1321 agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag 1381 aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc 1441 acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg 1501 ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc 1561 tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg 1621 tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca 1681 aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc 1741 tgcccggcga gaagaagaat ggcctgttcg gaaacctgat tgccctgagc ctgggcctga 1801 cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg 1861 acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc 1921 tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga 1981 acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc 2041 accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag 2101 agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc 2161 aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac 2221 tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca 2281 gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt 2341 tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc 2401 cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga 2461 gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc 2521 agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc 2581 ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat 2641 acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg 2701 tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact 2761 tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg 2821 cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca 2881 atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca 2941 gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga 3001 agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg 3061 gcatccggga caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg 3121 ccaacagaaa cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc 3181 agaaagccca ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg 3241 gcagccccgc cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga 3301 aagtgatggg ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga 3361 ccacccagaa gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca 3421 aagagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg 3481 agaagctgta cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg 3541 acatcaaccg gctgtccgac tacgatgtgg accatatcgt gcctcagagc tttctgaagg 3601 acgactccat cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca 3661 acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg 3721 ccaagctgat tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga 3781 gcgaactgga taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa 3841 agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc 3901 tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg 3961 atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc 4021 tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg 4081 tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa 4141 tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg 4201 agattaccct ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa 4261 ccggggagat cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca 4321 tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt 4381 ctatcctgcc caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta 4441 agaagtacgg cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag 4501 tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca 4561 tggaaagaag cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag 4621 aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg 4681 gccggaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc 4741 cctccaaata tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc 4801 ccgaggataa tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga 4861 tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca 4921 aagtgctgtc cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata 4981 tcatccacct gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca 5041 ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc 5101 accagagcat caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca 5161 aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa gaaaaagtaa gaattcctag 5221 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc 5281 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga 5341 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca 5401 ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggagc ggccgcagga 5461 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg 5521 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc 5581 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat 5641 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg 5701 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct 5761 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta 5821 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa 5881 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct 5941 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc 6001 aactctatct cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg 6061 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt 6121 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc 6181 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct 6241 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca 6301 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg 6361 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct 6421 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga 6481 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 6541 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 6601 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 6661 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact 6721 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc 6781 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 6841 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 6901 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 6961 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 7021 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 7081 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg 7141 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 7201 gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca 7261 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 7321 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 7381 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg 7441 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 7501 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 7561 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 7621 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 7681 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 7741 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 7801 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 7861 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 7921 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 7981 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 8041 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 8101 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 8161 ttcctggcct tttgctggcc ttttgctcac atgtgagggc ctatttccca tgattccttc 8221 atatttgcat atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa 8281 cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc 8341 agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt 8401 cgatttcttg gctttatata tcttgtggaa aggacgaaac accgtctgaa ccgtgtggga 8461 gccagttt //