LOCUS Exported 5138 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expresses sgRNA targeting mouse Idua intron 9 (sgIdua).. ACCESSION . VERSION . KEYWORDS pAAVsc.U6-sgIdua.CMV/CB-EGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5138) 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 5138) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5138 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 61..301 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 61..81 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 232..251 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 331..406 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 420..799 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 1095..1113 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" promoter 1118..1135 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 1136..1855 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 1136..1852 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1181..1202) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1442..1461) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1789..1810 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(1938..1957) /label=Bglob-pA-R /note="Rabbit beta-globin polyA region, reverse primer" polyA_signal 2003..2058 /label=-beta-globin poly(A) signal /note="rabbit beta-globin polyadenylation signal (Gil and Proudfoot, 1987)" repeat_region 2106..2235 /label=AAV2 ITR primer_bind complement(2254..2271) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(2254..2270) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2263..2285) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 2412..2867 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2499..2518) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2709..2730 /label=F1ori-F /note="F1 origin, forward primer" promoter 2893..2997 /gene="bla" /label=AmpR promoter CDS 2998..3858 /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(3216..3235) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4029..4617 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4518..4537 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4771..4788 /label=L4440 /note="L4440 vector, forward primer" protein_bind 4905..4926 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 4941..4971 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4979..4995 /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 4984..5006 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 5003..5019 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 5003..5019 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" ORIGIN 1 agcgagcgcg cagagaggga gtgtagccat gctctaggaa gatcaattca attcacgcgt 61 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 121 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 181 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 241 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 301 cgaaacaccg tgggagctag atattagggt gttttagagc tagaaatagc aagttaaaat 361 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttacgcgtcg 421 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc 481 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa 541 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac 601 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca 661 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg 721 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt 781 agtcatcgct attaccatgt cgaggccacg ttctgcttca ctctccccat ctcccccccc 841 tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg 901 gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc 961 ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga 1021 ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gcaagctcta 1081 gcctcgagaa tacttaatac gactcactat aggctagtaa tacgactcac tatagatggt 1141 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga 1201 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa 1261 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt 1321 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca 1381 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa 1441 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa 1501 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct 1561 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat 1621 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca 1681 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct 1741 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct 1801 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtaaagcgg 1861 ccctagcgtt taaacgggcc ctctagagtc gacccgggcg gcctcgagga cggggtgaac 1921 tacgcctgag gatccgatct ttttccctct gccaaaaatt atggggacat catgaagccc 1981 cttgagcatc tgacttctgg ctaataaagg aaatttattt tcattgcaat agtgtgttgg 2041 aattttttgt gtctctcact cggcctaggt agataagtag catggcgggt taatcattaa 2101 ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac 2161 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag 2221 cgagcgagcg cgcagcctta attaacctaa ttcactggcc gtcgttttac aacgtcgtga 2281 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag 2341 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa 2401 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 2461 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 2521 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 2581 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 2641 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 2701 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 2761 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 2821 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt 2881 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat 2941 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg 3001 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt 3061 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga 3121 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa 3181 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt 3241 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt 3301 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc 3361 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga 3421 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat 3481 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct 3541 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc 3601 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg 3661 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc 3721 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg 3781 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca 3841 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta 3901 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc 3961 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa 4021 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca 4081 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta 4141 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc 4201 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca 4261 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta 4321 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag 4381 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt 4441 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc 4501 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac 4561 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac 4621 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc 4681 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat 4741 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag 4801 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac 4861 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc 4921 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt 4981 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc cagatttaat 5041 taaggcctta attaggctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg 5101 gcgtcgggcg acctttggtc gcccggcctc agtgagcg //