LOCUS Exported 8505 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION One shot system to create CDK1as human cells. Encode Cas9 and guideRNA to inactivate human CDK1 gene.. ACCESSION . VERSION . KEYWORDS pX330_human CDK1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8505) AUTHORS Saldivar JC, Hamperl S, Bocek MJ, Chung M, Bass TE, Cisneros-Soberanis F, Samejima K, Xie L, Paulson JR, Earnshaw WC, Cortez D, Meyer T, Cimprich KA TITLE An intrinsic S/G2 checkpoint enforced by ATR. JOURNAL Science. 2018 Aug 24;361(6404):806-810. doi: 10.1126/science.aap9346. PUBMED 30139873 REFERENCE 2 (bases 1 to 8505) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8505 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 62..302 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 62..82 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 233..252 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 332..407 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 504..789 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 791..1065 /label=chicken beta-actin promoter intron 1066..1293 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 1305..1314 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1314..1379 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1386..1406 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1431..5531 /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 5532..5579 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5607..5624) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5613..5820 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5829..5969 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5829..5958 /label=AAV2 ITR rep_origin 6044..6499 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6131..6150) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6341..6362 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6516..6535) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6635..6657 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6695..6713) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6781..6885 /gene="bla" /label=AmpR promoter CDS 6886..7746 /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(7104..7123) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7917..8505 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8406..8425 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 61 tgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga 121 gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag 181 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca 241 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg 301 acgaaacacc gatttcccga attgcagtac tgttttagag ctagaaatag caagttaaaa 361 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttgttttag 421 agctagaaat agcaagttaa aataaggcta gtccgttttt agcgcgtgcg ccaattctgc 481 agacaaatgg ctctagaggt acccgttaca taacttacgg taaatggccc gcctggctga 541 ccgcccaacg acccccgccc attgacgtca atagtaacgc caatagggac tttccattga 601 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat 661 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattgtgcc 721 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct 781 attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc 841 ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg 901 gggggggggc gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg 961 agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg 1021 cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc 1081 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg 1141 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag 1201 ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac 1261 ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atggactata 1321 aggaccacga cggagactac aaggatcatg atattgatta caaagacgat gacgataaga 1381 tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt 1441 acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt 1501 acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga 1561 agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga 1621 agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga 1681 tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct 1741 tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg 1801 aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca 1861 gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc 1921 ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt 1981 tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg 2041 gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc 2101 tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga 2161 gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc 2221 agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc 2281 agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca 2341 tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat 2401 acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg 2461 agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg 2521 gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg 2581 gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct 2641 tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc 2701 ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga 2761 ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga 2821 tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg 2881 gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg 2941 agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga 3001 ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga 3061 aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga 3121 aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag 3181 atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg 3241 acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac 3301 tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg 3361 acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga 3421 agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt 3481 ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta 3541 aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg 3601 ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg 3661 acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca 3721 gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg 3781 aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc 3841 agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg 3901 accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga 3961 gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg 4021 gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc 4081 agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga 4141 gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc 4201 ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg 4261 agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg 4321 atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc 4381 acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg 4441 aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga 4501 gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact 4561 ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga 4621 caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga 4681 aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct 4741 tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg 4801 actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg 4861 tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg 4921 ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca 4981 agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg 5041 agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg 5101 aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc 5161 tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact 5221 acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg 5281 ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc 5341 aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca 5401 agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg 5461 ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc 5521 tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagt 5581 aagaattcct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt 5641 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc 5701 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg 5761 tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga 5821 gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct 5881 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt 5941 gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca 6001 tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc 6061 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc 6121 ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc 6181 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc 6241 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg 6301 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact 6361 ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt 6421 tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa 6481 atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag 6541 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc 6601 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt 6661 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag 6721 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg 6781 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga 6841 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat 6901 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca 6961 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc 7021 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca 7081 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg 7141 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca 7201 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata 7261 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag 7321 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg 7381 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca 7441 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta 7501 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct 7561 ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca 7621 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag 7681 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat 7741 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt 7801 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 7861 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 7921 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 7981 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 8041 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag 8101 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 8161 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 8221 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 8281 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 8341 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 8401 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 8461 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaa //