LOCUS Exported 8507 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION gRNA for targeting mouse Esrrb locus using CRISPR-cas technique. ACCESSION . VERSION . KEYWORDS pX330-Esrrb gRNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8507) AUTHORS Benchetrit H, Jaber M, Zayat V, Sebban S, Pushett A, Makedonski K, Zakheim Z, Radwan A, Maoz N, Lasry R, Renous N, Inbar M, Ram O, Kaplan T, Buganim Y TITLE Direct Induction of the Three Pre-implantation Blastocyst Cell Types from Fibroblasts. JOURNAL Cell Stem Cell. 2019 Jun 6;24(6):983-994.e7. doi: 10.1016/j.stem.2019.03.018. Epub 2019 Apr 25. PUBMED 31031139 REFERENCE 2 (bases 1 to 8507) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8507 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 55..295 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 55..75 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 226..245 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 324..399 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 496..781 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 783..1060 /label=chicken beta-actin promoter intron 1061..1288 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 1300..1309 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1309..1374 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1381..1401 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1426..5526 /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 5527..5574 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5602..5619) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5608..5815 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5824..5964 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5824..5953 /label=AAV2 ITR rep_origin 6039..6494 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6126..6145) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6336..6357 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6511..6530) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6630..6652 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6690..6708) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6776..6880 /gene="bla" /label=AmpR promoter CDS 6881..7741 /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(7099..7118) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7912..8500 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8401..8420 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgtgagggc 61 ctatttccca tgattccttc atatttgcat atacgataca aggctgttag agagataatt 121 ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa 181 taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt 241 accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac 301 accgggccca gcacatggac ggagttttag agctagaaat agcaagttaa aataaggcta 361 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttgtttt agagctagaa 421 atagcaagtt aaaataaggc tagtccgttt ttagcgcgtg cgccaattct gcagacaaat 481 ggctctagag gtacccgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa 541 cgacccccgc ccattgacgt caatagtaac gccaataggg actttccatt gacgtcaatg 601 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag 661 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattgtg cccagtacat 721 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat 781 ggtcgaggtg agccccacgt tctgcttcac tctccccatc tcccccccct ccccaccccc 841 aattttgtat ttatttattt tttaattatt ttgtgcagcg atgggggcgg gggggggggg 901 ggggcgcgcg ccaggcgggg gggggggggg gggggggggg gggggggcgg ggcggagagg 961 tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg 1021 gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg cgcgctgcct 1081 tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg ccccggctct gactgaccgc 1141 gttactccca caggtgagcg ggcgggacgg cccttctcct ccgggctgta attagctgag 1201 caagaggtaa gggtttaagg gatggttggt tggtggggta ttaatgttta attacctgga 1261 gcacctgcct gaaatcactt tttttcaggt tggaccggtg ccaccatgga ctataaggac 1321 cacgacggag actacaagga tcatgatatt gattacaaag acgatgacga taagatggcc 1381 ccaaagaaga agcggaaggt cggtatccac ggagtcccag cagccgacaa gaagtacagc 1441 atcggcctgg acatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag 1501 gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac 1561 ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga 1621 accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc 1681 agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg 1741 gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg 1801 gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc 1861 gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc 1921 cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc 1981 cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg 2041 gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc 2101 gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc cctgagcctg 2161 ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg 2221 agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac 2281 gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg 2341 agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac 2401 gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag 2461 tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga 2521 gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc 2581 gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac 2641 aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag 2701 gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc 2761 cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc 2821 agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct 2881 tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag 2941 gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa 3001 gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag 3061 gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag 3121 gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg 3181 ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc 3241 ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt 3301 gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa 3361 gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg 3421 atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac 3481 ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag 3541 gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat 3601 ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag 3661 ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag 3721 aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag 3781 ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg 3841 cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag 3901 gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc tcagagcttt 3961 ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag 4021 agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg 4081 ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc 4141 ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag 4201 atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat 4261 gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc 4321 cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac 4381 gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc 4441 gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag 4501 caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc 4561 aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac 4621 ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt gcggaaagtg 4681 ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc 4741 aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa gaaggactgg 4801 gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt gctggtggtg 4861 gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc 4921 accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc 4981 tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg 5041 gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg aaacgaactg 5101 gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag 5161 ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa gcactacctg 5221 gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat 5281 ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag agagcaggcc 5341 gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc cttcaagtac 5401 tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct ggacgccacc 5461 ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga 5521 ggcgacaaaa ggccggcggc cacgaaaaag gccggccagg caaaaaagaa aaagtaagaa 5581 ttcctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt 5641 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat 5701 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg 5761 tggggcagga cagcaagggg gaggattggg aagagaatag caggcatgct ggggagcggc 5821 cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg 5881 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg 5941 agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt 6001 gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt 6061 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc 6121 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca 6181 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc 6241 caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt 6301 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac 6361 aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt 6421 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt 6481 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag 6541 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc 6601 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc 6661 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa 6721 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg 6781 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata 6841 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg 6901 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac 6961 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact 7021 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat 7081 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga 7141 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac 7201 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat 7261 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac 7321 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct 7381 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac 7441 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga 7501 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg 7561 gtttattgct gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact 7621 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac 7681 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta 7741 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt 7801 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga 7861 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc 7921 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 7981 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 8041 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc 8101 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 8161 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 8221 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 8281 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc 8341 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 8401 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 8461 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgcca //