LOCUS Exported 8507 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION gRNA for targeting mouse Elf5 loci using CRISPR-cas technique. ACCESSION . VERSION . KEYWORDS pX330-Elf5 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 61..301 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 61..81 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 232..251 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 330..405 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 502..787 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 789..1066 /label=chicken beta-actin promoter intron 1067..1294 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 1306..1315 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1315..1380 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1387..1407 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1432..5532 /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 5533..5580 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5608..5625) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5614..5821 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5830..5970 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5830..5959 /label=AAV2 ITR rep_origin 6045..6500 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6132..6151) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6342..6363 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6517..6536) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6636..6658 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6696..6714) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6782..6886 /gene="bla" /label=AmpR promoter CDS 6887..7747 /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(7105..7124) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7918..8506 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8407..8426 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 61 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 121 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 181 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 241 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 301 cgaaacaccg ctgatggaca ccggacaccg ttttagagct agaaatagca agttaaaata 361 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag 421 ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag 481 acaaatggct ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc 541 gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg 601 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 661 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca 721 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 781 taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc 841 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg 901 gggggggggg cgcgcgccrg gggggggggg ggggggsggg gggsggggsg gggsgaggcg 961 gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag 1021 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg 1081 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact 1141 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta 1201 gctgagcaag aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta 1261 cctggagcac ctgcctgaaa tcactttttt tcaggttgga ccggtgccac catggactat 1321 aaggaccacg acggagacta caaggatcat gatattgatt acaaagacga tgacgataag 1381 atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc cgacaagaag 1441 tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag 1501 tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag 1561 aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg 1621 aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag 1681 atcttcagca acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc 1741 ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac 1801 gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac 1861 agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc 1921 cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg 1981 ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc 2041 ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat 2101 ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg 2161 agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg 2221 cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac 2281 cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac 2341 atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga 2401 tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct 2461 gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac 2521 ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac 2581 ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc 2641 ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg 2701 cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg 2761 accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg 2821 atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag 2881 ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac 2941 gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg 3001 accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag 3061 aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg 3121 aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa 3181 gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag 3241 gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca 3301 ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac 3361 gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg 3421 aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag 3481 tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt 3541 aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt 3601 gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg 3661 gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc 3721 agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc 3781 gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc 3841 cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg 3901 gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag 3961 agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg 4021 ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg 4081 cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag 4141 agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc 4201 cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac 4261 gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc 4321 gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc 4381 cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg 4441 gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag 4501 agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac 4561 tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag 4621 acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg 4681 aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc 4741 ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag 4801 gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg 4861 gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg 4921 gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc 4981 aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc 5041 gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac 5101 gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag 5161 ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac 5221 tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac 5281 gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag 5341 caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc 5401 aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac 5461 gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag 5521 ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag 5581 taagaattcc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg 5641 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt 5701 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg 5761 gtggggtggg gcaggacagc aagggggagg attgggaaga gaatagcagg catgctgggg 5821 agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc 5881 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag 5941 tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc 6001 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg 6061 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc 6121 cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc 6181 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct 6241 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac 6301 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac 6361 tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat 6421 ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa 6481 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata 6541 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct 6601 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt 6661 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata 6721 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt 6781 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag 6841 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca 6901 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc 6961 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat 7021 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc 7081 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg 7141 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc 7201 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat 7261 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga 7321 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc 7381 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc 7441 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt 7501 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc 7561 tggctggttt attgctgata aatctggagc cggtgagcgt ggaagccgcg gtatcattgc 7621 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca 7681 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca 7741 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt 7801 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta 7861 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg 7921 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 7981 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 8041 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa 8101 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 8161 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 8221 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 8281 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag 8341 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 8401 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 8461 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaa //