LOCUS Exported 4886 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses sgRNA targeting the intron in the MADM alleles (GT and TG).. ACCESSION . VERSION . KEYWORDS pAAVsc.U6-sgGTTG.CMV/CB-Gluc SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4886) AUTHORS Wang D, Li J, Song CQ, Tran K, Mou H, Wu PH, Tai PWL, Mendonca CA, Ren L, Wang BY, Su Q, Gessler DJ, Zamore PD, Xue W, Gao G TITLE Cas9-mediated allelic exchange repairs compound heterozygous recessive mutations in mice. JOURNAL Nat Biotechnol. 2018 Oct;36(9):839-842. doi: 10.1038/nbt.4219. Epub 2018 Aug 13. PUBMED 30102296 REFERENCE 2 (bases 1 to 4886) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4886 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 136..155 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 234..309 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 323..702 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" regulatory 992..1001 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 998..1555 /codon_start=1 /product="secreted Gaussia luciferase" /label=hGLuc /note="human codon-optimized" /translation="MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRG KLPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESA QGGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQ RCATFASKIQGQVDKIKGAGGD" primer_bind complement(1591..1610) /label=Bglob-pA-R /note="Rabbit beta-globin polyA region, reverse primer" polyA_signal 1656..1711 /label=-beta-globin poly(A) signal /note="rabbit beta-globin polyadenylation signal (Gil and Proudfoot, 1987)" repeat_region 1759..1888 /label=AAV2 ITR primer_bind complement(1907..1924) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(1907..1923) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1916..1938) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 2065..2520 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2152..2171) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2362..2383 /label=F1ori-F /note="F1 origin, forward primer" promoter 2546..2650 /gene="bla" /label=AmpR promoter CDS 2651..3511 /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(2869..2888) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 3682..4270 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4171..4190 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4424..4441 /label=L4440 /note="L4440 vector, forward primer" protein_bind 4558..4579 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 4594..4624 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4632..4648 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 4637..4659 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 4656..4672 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 4656..4672 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" promoter 4851..205 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 4851..4871 /label=hU6-F /note="Human U6 promoter, forward primer" ORIGIN 1 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata 61 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa 121 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg 181 gctttatata tcttgtggaa aggacgaaac accgggtggg aaaatagacc aatgttttag 241 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg 301 agtcggtgct tttttacgcg tcgacattga ttattgacta gttattaata gtaatcaatt 361 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat 421 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt 481 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa 541 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc 601 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct 661 acttggcagt acatctacgt attagtcatc gctattacca tgtcgaggcc acgttctgct 721 tcactctccc catctccccc ccctccccac ccccaatttt gtatttattt attttttaat 781 tattttgtgc agcgatgggg gcgggggggg ggggcgcgcg ccaggcgggg cggggcgggg 841 cgaggggcgg ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct 901 ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc 961 gcggcgggcg ggagcaagct ctagcctcga ggccaccatg ggagtcaaag ttctgtttgc 1021 cctgatctgc atcgctgtgg ccgaggccaa gcccaccgag aacaacgagg acttcaacat 1081 cgtggccgtg gccagcaact tcgcgaccac ggatctcgat gctgaccgcg ggaagttgcc 1141 cggcaagaag ctgccgctgg aggtgctcaa agagatggaa gccaatgccc ggaaagctgg 1201 ctgcaccagg ggctgtctga tctgcctgtc ccacatcaag tgcacgccca agatgaagaa 1261 gttcatccca ggacgctgcc acacctacga aggcgacaaa gagtccgcac agggcggcat 1321 aggcgaggcg atcgtcgaca ttcctgagat tcctgggttc aaggacttgg agcccatgga 1381 gcagttcatc gcacaggtcg atctgtgtgt ggactgcaca actggctgcc tcaaagggct 1441 tgccaacgtg cagtgttctg acctgctcaa gaagtggctg ccgcaacgct gtgcgacctt 1501 tgccagcaag atccagggcc aggtggacaa gatcaagggg gccggtggtg actagctcga 1561 ggacggggtg aactacgcct gaggatccga tctttttccc tctgccaaaa attatgggga 1621 catcatgaag ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc 1681 aatagtgtgt tggaattttt tgtgtctctc actcggccta ggtagataag tagcatggcg 1741 ggttaatcat taactacaag gaacccctag tgatggagtt ggccactccc tctctgcgcg 1801 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg 1861 cggcctcagt gagcgagcga gcgcgcagcc ttaattaacc taattcactg gccgtcgttt 1921 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc 1981 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt 2041 tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg 2101 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 2161 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg 2221 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 2281 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 2341 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta 2401 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 2461 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt 2521 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca 2581 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa 2641 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt 2701 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca 2761 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 2821 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc 2881 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca 2941 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt 3001 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct 3061 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt 3121 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga 3181 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact 3241 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc 3301 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga 3361 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt 3421 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga 3481 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact 3541 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga 3601 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 3661 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 3721 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 3781 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta 3841 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 3901 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 3961 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 4021 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 4081 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 4141 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 4201 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 4261 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 4321 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt 4381 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga 4441 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta 4501 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa 4561 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat 4621 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta 4681 cgccagattt aattaaggcc ttaattaggc tgcgcgctcg ctcgctcact gaggccgccc 4741 gggcaaagcc cgggcgsscy crggcaaagc ccgggcggcc tcagtgagcg agcgagcgcg 4801 cagagaggga gtgtagccat gctctaggaa gatcaattca attcacgcgt gagggcctat 4861 ttcccatgat tccttcatat ttgcat //