LOCUS Exported 8436 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION For transient expression of spCas9-nickase and one sgRNA targeting the mouse CTCF locus. Use together with pX335-EN477.. ACCESSION . VERSION . KEYWORDS pX335-EN475 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8436) AUTHORS Nora EP, Goloborodko A, Valton AL, Gibcus JH, Uebersohn A, Abdennur N, Dekker J, Mirny LA, Bruneau BG TITLE Targeted Degradation of CTCF Decouples Local Insulation of Chromosome Domains from Genomic Compartmentalization. JOURNAL Cell. 2017 May 18;169(5):930-944.e22. doi: 10.1016/j.cell.2017.05.004. PUBMED 28525758 REFERENCE 2 (bases 1 to 8436) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8436 /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 271..346 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 443..728 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 730..1007 /label=chicken beta-actin promoter intron 1008..1235 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" CDS 1256..1282 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 1286..1306 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1316..5416 /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 5420..5440 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" primer_bind complement(5477..5494) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5483..5690 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5699..5839 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5699..5828 /label=AAV2 ITR rep_origin 5914..6369 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6001..6020) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6211..6232 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6386..6405) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6505..6527 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6565..6583) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6651..6755 /gene="bla" /label=AmpR promoter CDS 6756..7616 /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(6974..6993) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7787..8375 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8276..8295 /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 atcaccggtc catcatgctg gttttagagc tagaaatagc aagttaaaat 301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga 361 gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca 421 gacaaatggc tctagaggta cccgttacat aacttacggt aaatggcccg cctggctgac 481 cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac 541 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata 601 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc 661 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta 721 ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc 781 cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg 841 gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc 901 ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga 961 ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc 1021 gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac 1081 tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt 1141 agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt 1201 acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatgtaccc 1261 atacgatgtt ccagattacg cttcgccgaa gaaaaagcgc aaggtcgaag cgtccgacaa 1321 gaagtacagc atcggcctgg ccatcggcac caactctgtg ggctgggccg tgatcaccga 1381 cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat 1441 caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg 1501 gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca 1561 agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga 1621 gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt 1681 ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt 1741 ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa 1801 gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa 1861 gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc 1921 cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga 1981 aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggca acctgattgc 2041 cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa 2101 actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg 2161 cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag 2221 cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa 2281 gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct 2341 gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat 2401 tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat 2461 ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg 2521 gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct 2581 gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat 2641 cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc 2701 ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga 2761 caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc 2821 caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga 2881 gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga 2941 gcagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca 3001 gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt 3061 ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga 3121 caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct 3181 gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt 3241 cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag 3301 ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct 3361 gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac 3421 ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca 3481 cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt 3541 ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat 3601 ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg 3661 gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa 3721 cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta 3781 cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc 3841 tcagagcttt ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa 3901 ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg 3961 gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc 4021 cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga 4081 aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta 4141 cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt 4201 gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca 4261 cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa 4321 gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc 4381 caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat 4441 gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat 4501 cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt 4561 gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg 4621 cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa 4681 gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt 4741 gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct 4801 gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga 4861 agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct 4921 gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg 4981 aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga 5041 gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa 5101 gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc 5161 cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag 5221 agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc 5281 cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct 5341 ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc 5401 tcagctggga ggcgacagcc ccaagaagaa gagaaaggtg gaggccagct aagaattcct 5461 agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc 5521 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat 5581 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg 5641 caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga gcggccgcag 5701 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc 5761 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga 5821 gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt 5881 atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg 5941 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg 6001 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 6061 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 6121 aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 6181 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 6241 tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt 6301 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt 6361 ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc 6421 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg 6481 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat 6541 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca 6601 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc 6661 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct 6721 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg 6781 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg 6841 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc 6901 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca 6961 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac 7021 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa 7081 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg 7141 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt 7201 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg 7261 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc 7321 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga 7381 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta 7441 ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc 7501 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg 7561 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt 7621 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa 7681 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt 7741 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt 7801 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt 7861 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga 7921 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag 7981 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata 8041 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg 8101 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga 8161 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca 8221 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa 8281 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt 8341 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac 8401 ggttcctggc cttttgctgg ccttttgctc acatgt //