LOCUS Exported 8435 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Encodes an sgRNA that creates a SSB near the poly(A) site of human NEAT1_1 isoform.. ACCESSION . VERSION . KEYWORDS pX335-NEAT1_IS_v1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8435) AUTHORS Li R, Harvey AR, Hodgetts SI, Fox AH TITLE Functional dissection of NEAT1 using genome editing reveals substantial localisation of the NEAT1_1 isoform outside paraspeckles. JOURNAL RNA. 2017 Mar 21. pii: rna.059477.116. doi: 10.1261/rna.059477.116. PUBMED 28325845 REFERENCE 2 (bases 1 to 8435) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8435 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..241 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1..21 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 172..191 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 270..345 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 442..727 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 729..1006 /label=chicken beta-actin promoter intron 1007..1234 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" CDS 1255..1281 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 1285..1305 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1315..5415 /codon_start=1 /product="nickase mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9(D10A) /note="generates RNA-guided single strand nicks in DNA due to the D10A mutation in the RuvC catalytic domain" /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 5419..5439 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" primer_bind complement(5476..5493) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5482..5689 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5698..5838 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5698..5827 /label=AAV2 ITR rep_origin 5913..6368 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6000..6019) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6210..6231 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6385..6404) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6504..6526 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6564..6582) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6650..6754 /gene="bla" /label=AmpR promoter CDS 6755..7615 /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(6973..6992) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7786..8374 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8275..8294 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 241 cgaaacaccg tattagtcac gcatgtatgg ttttagagct agaaatagca agttaaaata 301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag 361 ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag 421 acaaatggct ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc 481 gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg 541 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 601 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca 661 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 721 taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc 781 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg 841 gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg 901 gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag 961 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg 1021 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact 1081 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta 1141 gctgagcaag aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta 1201 cctggagcac ctgcctgaaa tcactttttt tcaggttgga ccggtgccac catgtaccca 1261 tacgatgttc cagattacgc ttcgccgaag aaaaagcgca aggtcgaagc gtccgacaag 1321 aagtacagca tcggcctggc catcggcacc aactctgtgg gctgggccgt gatcaccgac 1381 gagtacaagg tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc 1441 aagaagaacc tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg 1501 ctgaagagaa ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa 1561 gagatcttca gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag 1621 tccttcctgg tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg 1681 gacgaggtgg cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg 1741 gacagcaccg acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag 1801 ttccggggcc acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag 1861 ctgttcatcc agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc 1921 agcggcgtgg acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa 1981 aatctgatcg cccagctgcc cggcgagaag aagaatggcc tgttcggcaa cctgattgcc 2041 ctgagcctgg gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa 2101 ctgcagctga gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc 2161 gaccagtacg ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc 2221 gacatcctga gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag 2281 agatacgacg agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg 2341 cctgagaagt acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt 2401 gacggcggag ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg 2461 gacggcaccg aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg 2521 accttcgaca acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg 2581 cggcggcagg aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc 2641 ctgaccttcc gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc 2701 tggatgacca gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac 2761 aagggcgctt ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc 2821 aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag 2881 ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag 2941 cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag 3001 ctgaaagagg actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg 3061 gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac 3121 aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg 3181 acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc 3241 gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc 3301 cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg 3361 aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc 3421 tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac 3481 attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg 3541 gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg 3601 gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg 3661 atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac 3721 acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac 3781 gtggaccagg aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct 3841 cagagctttc tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac 3901 cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg 3961 cggcagctgc tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc 4021 gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa 4081 acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac 4141 gacgagaatg acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg 4201 tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac 4261 gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag 4321 ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc 4381 aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg 4441 aactttttca agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc 4501 gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg 4561 cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc 4621 ggcttcagca aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag 4681 aaggactggg accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg 4741 ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg 4801 ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa 4861 gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg 4921 ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga 4981 aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag 5041 aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag 5101 cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc 5161 gacgctaatc tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga 5221 gagcaggccg agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc 5281 ttcaagtact ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg 5341 gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct 5401 cagctgggag gcgacagccc caagaagaag agaaaggtgg aggccagcta agaattccta 5461 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct 5521 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg 5581 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc 5641 aggacagcaa gggggaggat tgggaagaga atagcaggca tgctggggag cggccgcagg 5701 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg 5761 ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag 5821 cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta 5881 tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc 5941 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc 6001 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct 6061 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa 6121 acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc 6181 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact 6241 caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg 6301 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt 6361 tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc 6421 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc 6481 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc 6541 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat 6601 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc 6661 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 6721 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc 6781 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt 6841 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct 6901 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac 6961 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact 7021 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa 7081 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga 7141 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt 7201 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga 7261 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg 7321 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat 7381 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat 7441 tgctgataaa tctggagccg gtgagcgtgg aagccgcggt atcattgcag cactggggcc 7501 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga 7561 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc 7621 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag 7681 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc 7741 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 7801 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 7861 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 7921 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 7981 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 8041 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 8101 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 8161 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 8221 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 8281 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 8341 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 8401 gttcctggcc ttttgctggc cttttgctca catgt //