LOCUS Exported 8487 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION gRNA/Cas9 expression for 2nd editing using pSLHDR04. ACCESSION . VERSION . KEYWORDS pSLHDR_R4 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8487) AUTHORS Ikeda K, Uchida N, Nishimura T, White J, Martin RM, Nakauchi H, Sebastiano V, Weinberg KI, Porteus MH TITLE Efficient scarless genome editing in human pluripotent stem cells. JOURNAL Nat Methods. 2018 Dec;15(12):1045-1047. doi: 10.1038/s41592-018-0212-y. Epub 2018 Nov 30. PUBMED 30504872 REFERENCE 2 (bases 1 to 8487) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8487 /organism="synthetic DNA construct" /mol_type="other DNA" intron 187..414 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 426..435 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 435..500 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 507..527 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 552..4652 /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 4653..4700 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(4728..4745) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 4734..4941 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 4950..5090 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4950..5079 /label=AAV2 ITR rep_origin 5165..5620 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5252..5271) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5462..5483 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5637..5656) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5756..5778 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5816..5834) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5902..6006 /gene="bla" /label=AmpR promoter CDS 6007..6867 /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(6225..6244) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7038..7626 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7527..7546 /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 7688..7928 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 7688..7708 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 7859..7878 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 7958..8033 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 8130..8415 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" ORIGIN 1 tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggg gcggggcgrg 61 ggggggggsg gggggggggg ggggggkgcg gcggcagcca atcagagcgg cgcgctccga 121 aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg 181 cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc 241 cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct 301 tctcctccgg gctgtaatta gctgagcaag aggtaagggt ttaagggatg gttggttggt 361 ggggtattaa tgtttaatta cctggagcac ctgcctgaaa tcactttttt tcaggttgga 421 ccggtgccac catggactat aaggaccacg acggagacta caaggatcat gatattgatt 481 acaaagacga tgacgataag atggccccaa agaagaagcg gaaggtcggt atccacggag 541 tcccagcagc cgacaagaag tacagcatcg gcctggacat cggcaccaac tctgtgggct 601 gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca 661 ccgaccggca cagcatcaag aagaacctga tcggagccct gctgttcgac agcggcgaaa 721 cagccgaggc cacccggctg aagagaaccg ccagaagaag atacaccaga cggaagaacc 781 ggatctgcta tctgcaagag atcttcagca acgagatggc caaggtggac gacagcttct 841 tccacagact ggaagagtcc ttcctggtgg aagaggataa gaagcacgag cggcacccca 901 tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc atctaccacc 961 tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc tatctggccc 1021 tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg aaccccgaca 1081 acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg 1141 aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc agactgagca 1201 agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag aatggcctgt 1261 tcggaaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc aacttcgacc 1321 tggccgagga tgccaaactg cagctgagca aggacaccta cgacgacgac ctggacaacc 1381 tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag aacctgtccg 1441 acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag gcccccctga 1501 gcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg ctgaaagctc 1561 tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag agcaagaacg 1621 gctacgccgg ctacattgac ggcggagcca gccaggaaga gttctacaag ttcatcaagc 1681 ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac agagaggacc 1741 tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc ccaccagatc cacctgggag 1801 agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag gacaaccggg 1861 aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct ctggccaggg 1921 gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc ccctggaact 1981 tcgaggaagt ggtggacaag ggcgcttccg cccagagctt catcgagcgg atgaccaact 2041 tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact 2101 tcaccgtgta taacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg 2161 ccttcctgag cggcgagcag aaaaaggcca tcgtggacct gctgttcaag accaaccgga 2221 aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg 2281 tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc 2341 tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag 2401 atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa 2461 cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg 2521 gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag tccggcaaga 2581 caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg cagctgatcc 2641 acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg 2701 atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcc 2761 tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga 2821 acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc 2881 gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag 2941 aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga 3001 atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg 3061 tggaccatat cgtgcctcag agctttctga aggacgactc catcgacaac aaggtgctga 3121 ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga 3181 agatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag agaaagttcg 3241 acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca 3301 agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc ctggactccc 3361 ggatgaacac taagtacgac gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc 3421 tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga 3481 tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga accgccctga 3541 tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg 3601 tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct 3661 tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc 3721 ggaagcggcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg gataagggcc 3781 gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga 3841 ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgata 3901 agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca 3961 ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga 4021 agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc 4081 ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc 4141 tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg 4201 gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc 4261 tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt 4321 ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc gagttctcca 4381 agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcacc 4441 gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc 4501 tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca 4561 gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga 4621 cacggatcga cctgtctcag ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg 4681 gccaggcaaa aaagaaaaag taagaattcc tagagctcgc tgatcagcct cgactgtgcc 4741 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg 4801 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag 4861 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga 4921 gaatagcagg catgctgggg agcggccgca ggaaccccta gtgatggagt tggccactcc 4981 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg 5041 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat 5101 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca 5161 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg 5221 accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc 5281 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga 5341 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt 5401 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat 5461 agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat 5521 ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa 5581 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca 5641 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg 5701 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg 5761 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc 5821 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt 5881 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca 5941 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 6001 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc 6061 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg 6121 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt 6181 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta 6241 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat 6301 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga 6361 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca 6421 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact 6481 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc 6541 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact 6601 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 6661 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 6721 ggaagccgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 6781 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata 6841 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag 6901 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat 6961 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 7021 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 7081 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 7141 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg 7201 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 7261 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 7321 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 7381 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 7441 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 7501 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 7561 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 7621 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 7681 cacatgtgag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt 7741 tagagagata attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg 7801 acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga 7861 ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg 7921 gaaaggacga aacaccgacg gtctccgatc gatgtctgtt ttagagctag aaatagcaag 7981 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg 8041 ttttagagct agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa 8101 ttctgcagac aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct 8161 ggctgaccgc ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc 8221 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg 8281 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat 8341 tgtgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc 8401 atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc 8461 ccctccccac ccccaatttt gtattta //