LOCUS Exported 6768 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION AAV-CRISPR library for pool mutagenesis of top tumor suppressor genes. ACCESSION . VERSION . KEYWORDS EFS-mTSG SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6768) AUTHORS Wang G, Chow RD, Ye L, Guzman CD, Dai X, Dong MB, Zhang F, Sharp PA, Platt RJ, Chen S TITLE Mapping a functional cancer genome atlas of tumor suppressors in mouse liver using AAV-CRISPR-mediated direct in vivo screening. JOURNAL Sci Adv. 2018 Feb 28;4(2):eaao5508. doi: 10.1126/sciadv.aao5508. eCollection 2018 Feb. PUBMED 29503867 REFERENCE 2 (bases 1 to 6768) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6768 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..141 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 12..141 /label=AAV2 ITR promoter 156..396 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" misc_RNA 425..500 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 514..754 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" gap 763..782 /estimated_length=20 misc_RNA 783..858 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 913..1124 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" regulatory 1132..1141 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1138..2787 /codon_start=1 /gene="luc+" /product="firefly luciferase" /label=luciferase /note="enhanced luc+ version of the luciferase gene" /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV" primer_bind complement(1204..1222) /label=LucNrev /note="Firefly luciferase, reverse primer" CDS 2803..2859 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 2860..2880 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3907..3933 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" polyA_signal 3955..4003 /note="synthetic polyadenylation signal" repeat_region 4031..4171 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4031..4160 /label=AAV2 ITR rep_origin 4246..4701 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4333..4352) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4543..4564 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4718..4737) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4837..4859 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(4897..4915) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 4983..5087 /gene="bla" /label=AmpR promoter CDS 5088..5948 /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(5306..5325) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6119..6707 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6608..6627 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 121 actccatcac taggggttcc tgcggccgca cgcgtgaggg cctatttccc atgattcctt 181 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa 241 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg 301 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt 361 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgtgtaa tagctcctgc 421 atgggtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa 481 aagtggcacc gagtcggtgc tttttttcta gaagagggcc tatttcccat gattccttca 541 tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac 601 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca 661 gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc 721 gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccnnnnnnnn nnnnnnnnnn 781 nngttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa 841 gtggcaccga gtcggtgctt ttttggtacc aggtcttgaa aggagtggga attggctccg 901 gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg 961 tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg 1021 tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg 1081 ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggcgtac ggccaccatg 1141 gaagacgcca aaaacataaa gaaaggcccg gcgccattct atccgctgga agatggaacc 1201 gctggagagc aactgcataa ggctatgaag agatacgccc tggttcctgg aacaattgct 1261 tttacagatg cacatatcga ggtggacatc acttacgctg agtacttcga aatgtccgtt 1321 cggttggcag aagctatgaa acgatatggg ctgaatacaa atcacagaat cgtcgtatgc 1381 agtgaaaact ctcttcaatt ctttatgccg gtgttgggcg cgttatttat cggagttgca 1441 gttgcgcccg cgaacgacat ttataatgaa cgtgaattgc tcaacagtat gggcatttcg 1501 cagcctaccg tggtgttcgt ttccaaaaag gggttgcaaa aaattttgaa cgtgcaaaaa 1561 aagctcccaa tcatccaaaa aattattatc atggattcta aaacggatta ccagggattt 1621 cagtcgatgt acacgttcgt cacatctcat ctacctcccg gttttaatga atacgatttt 1681 gtgccagagt ccttcgatag ggacaagaca attgcactga tcatgaactc ctctggatct 1741 actggtctgc ctaaaggtgt cgctctgcct catagaactg cctgcgtgag attctcgcat 1801 gccagagatc ctatttttgg caatcaaatc attccggata ctgcgatttt aagtgttgtt 1861 ccattccatc acggttttgg aatgtttact acactcggat atttgatatg tggatttcga 1921 gtcgtcttaa tgtatagatt tgaagaggag ctgtttctga ggagccttca ggattacaag 1981 attcaaagtg cgctgctggt gccaacccta ttctccttct tcgccaaaag cactctgatt 2041 gacaaatacg atttatctaa tttacacgaa attgcttctg gtggcgctcc cctctctaag 2101 gaagtcgggg aagcggttgc caagaggttc catctgccag gtatcaggca aggatatggg 2161 ctcactgaga ctacatcagc tattctgatt acacccgagg gggatgataa accgggcgcg 2221 gtcggtaaag ttgttccatt ttttgaagcg aaggttgtgg atctggatac cgggaaaacg 2281 ctgggcgtta atcaaagagg cgaactgtgt gtgagaggtc ctatgattat gtccggttat 2341 gtaaacaatc cggaagcgac caacgccttg attgacaagg atggatggct acattctgga 2401 gacatagctt actgggacga agacgaacac ttcttcatcg ttgaccgcct gaagtctctg 2461 attaagtaca aaggctatca ggtggctccc gctgaattgg aatccatctt gctccaacac 2521 cccaacatct tcgacgcagg tgtcgcaggt cttcccgacg atgacgccgg tgaacttccc 2581 gccgccgttg ttgttttgga gcacggaaag acgatgacgg aaaaagagat cgtggattac 2641 gtcgccagtc aagtaacaac cgcgaaaaag ttgcgcggag gagttgtgtt tgtggacgaa 2701 gtaccgaaag gtcttaccgg aaaactcgac gcaagaaaaa tcagagagat cctcataaag 2761 gccaagaagg gcggaaagat cgccgtggct agcggaagcg gagccactaa cttctccctg 2821 ttgaaacaag caggggatgt cgaagagaat cccgggccac ccaagaagaa gaggaaggtg 2881 tccaatctcc tgactgttca ccagaacctc cctgcgctgc cagtagatgc cactagcgat 2941 gaggtcagga aaaatctcat ggatatgttt agggatagac aggcgttttc tgaacacacc 3001 tggaaaatgc tgcttagcgt gtgccgatcc tgggcagcct ggtgtaagct gaacaatcgc 3061 aaatggttcc ccgccgagcc ggaggacgtg cgcgattacc tgctgtatct ccaggcaaga 3121 gggctggctg tcaagactat ccagcagcac ttgggccaac tgaatatgct gcatcgacgc 3181 agcgggctcc cccggcctag cgattcaaac gcagtctccc ttgttatgag gagaattaga 3241 aaggaaaacg tagatgcggg tgagagggct aagcaggctc tcgcttttga gcggactgat 3301 ttcgaccagg tcagatccct gatggagaac agcgatcggt gccaggacat caggaacctc 3361 gcatttctgg gaattgcata taacacactt ctgcgcatag ctgagatcgc ccggatcaga 3421 gtgaaagaca tcagtcgaac ggacggcggc cggatgctta ttcatattgg acgcacaaag 3481 acattggtca gcaccgctgg cgttgaaaag gccttgtccc tgggcgtaac gaagctggtg 3541 gaaagatgga tctcagtgtc cggcgtggct gacgacccta ataattactt gttctgtcga 3601 gtgagaaaaa acggagtcgc cgcgccctct gccaccagcc aattgagtac acgggccctt 3661 gaagggatct ttgaggcaac ccaccgactc atatacggag ccaaggatga cagtggccag 3721 aggtatctcg cctggtcagg tcattctgct agggtggggg ccgcacgaga catggcgcgg 3781 gcaggagtct ccataccaga gattatgcaa gctggaggtt ggacaaatgt gaacatcgtt 3841 atgaactata tccgcaatct tgactctgaa accggggcca tggtgagact gctcgaagat 3901 ggtgactacc catacgatgt tccagattac gcttaagaat tcgatatcaa gcttaataaa 3961 agatctttat tttcattaga tctgtgtgtt ggttttttgt gtggtaacca cgtgcggacc 4021 gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 4081 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 4141 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg 4201 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg 4261 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg 4321 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc 4381 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc 4441 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga 4501 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa 4561 ctggaacaac actcaaccct atctcgggct attcttttga tttataaggg attttgccga 4621 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca 4681 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat 4741 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 4801 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt 4861 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat 4921 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg 4981 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 5041 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 5101 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc 5161 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca 5221 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc 5281 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg 5341 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac 5401 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca 5461 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg 5521 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 5581 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg 5641 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat 5701 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 5761 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg 5821 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 5881 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 5941 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt 6001 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt 6061 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 6121 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 6181 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca 6241 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca 6301 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 6361 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 6421 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 6481 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 6541 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 6601 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 6661 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 6721 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt //