LOCUS Exported 6368 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION expression of gRNA under control of the Drosophila U6:3 promoter. ACCESSION . VERSION . KEYWORDS pCFD3-dU6:3gRNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6368) AUTHORS Port F, Chen HM, Lee T, Bullock SL TITLE Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. JOURNAL Proc Natl Acad Sci U S A. 2014 Jul 7. pii: 201405500. PUBMED 25002478 REFERENCE 2 (bases 1 to 6368) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6368 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(17..37) /label=T3 /note="T3 promoter, forward primer" promoter complement(17..35) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(56..72) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(56..72) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(69..91) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 80..96 /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)." promoter complement(104..134) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 149..170 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(287..304) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(458..1046) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(538..557) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(1217..2077) /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 1840..1859 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(2078..2182) /gene="bla" /label=AmpR promoter rep_origin 2209..2665 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2296..2315) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2506..2527 /label=F1ori-F /note="F1 origin, forward primer" primer_bind 2791..2813 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 2805..2822 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 2806..2822 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 2832..2851 /label=T7 /note="T7 promoter, forward primer" promoter 2832..2850 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS complement(join(3042..3159,3236..3329,3421..4027,4082..4215, 4276..4436,4510..4535)) /codon_start=1 /product="tryptophan oxygenase" /label=vermilion /note="selectable marker, required to synthesize the brown eye pigment in Drosophila" /translation="MSCPYAGNGNDHDDSAVPLTTEVGKIYGEYLMLDKLLDAQCMLSE EDKRPVHDEHLFIITHQAYELWFKQIIFEFDSIRDMLDAEVIDETKTLEIVKRLNRVVL ILKLLVDQVPILETMTPLDFMDFRKYLAPASGFQSLQFRLIENKLGVLTEQRVRYNQKY SDVFSDEEARNSIRNSEKDPSLLELVQRWLERTPGLEESGFNFWAKFQESVDRFLEAQV QSAMEEPVEKAKNYRLMDIEKRREVYRSIFDPAVHDALVRRGDRRFSHRALQGAIMITF YRDEPRFSQPHQLLTLLMDIDSLITKWRYNHVIMVQRMIGSQQLGTGGSSGYQYLRSTL SDRYKVFLDLFNLSTFLIPREAIPPLDETIRKKLINKSV" protein_bind 4850..4919 /label=attB /bound_moiety="phage phi-C31 integrase" /note="attB site for the phi-C31 integrase (Groth et al., 2000)" protein_bind 5161..5546 /label=gypsy insulator /bound_moiety="Su(Hw), CP190, and Mod(mdg4)67.2" /note="chromatin insulator from Drosophila" promoter 5560..5980 /label=dU6-3 promoter /note="RNA polymerase III promoter for Drosophila U6-3 snRNA (Port et al., 2014)" misc_RNA 5999..6074 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" ORIGIN 1 gctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat 61 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa 121 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc 181 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc 241 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact 301 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 361 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 421 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 481 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 541 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 601 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac 661 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 721 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 781 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 841 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa 901 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 961 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 1021 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 1081 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 1141 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 1201 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 1261 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 1321 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 1381 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 1441 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 1501 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 1561 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 1621 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 1681 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 1741 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 1801 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca 1861 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 1921 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 1981 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa 2041 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt 2101 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 2161 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gcgccctgta 2221 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca 2281 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct 2341 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc 2401 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat 2461 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc 2521 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc 2581 cgatttcggc ctattggtta aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt 2641 aacaaaatat taacgcttac aatttccatt cgccattcag gctgcgcaac tgttgggaag 2701 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa 2761 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca 2821 gtgagcgcgc gtaatacgac tcactatagg gcgaattggg tacaagcttg gatttatttt 2881 gttatgttat atgtattata tgtcagacat aaagaaaagg aacacatcaa atgtgataac 2941 aaagactaaa caagtaattt tattacacca aaacgacaaa acagtaggca gaacaaacaa 3001 cgcatagcca aacattgacg aattggatac cctgccgatt gtcagacact tttgttgatc 3061 agtttcttgc gaatggtctc gtccagcggt ggaatcgcct cgcggggaat cagaaaagtg 3121 gacagattga acagatccag aaacaccttg taccgatcac tgaaaccaaa aaaaaacaaa 3181 gggagaacag tttgagttca ttgatccccg atataatcac atctgcgatg atcacctgag 3241 agtggagcgc agatattgat atccagacga gccaccagtg cccaactgtt gggatccaat 3301 catgcgttgc accatgatca cgtgattgtc tgcggcggga atagaaagta tttggttagg 3361 aaaaccagtc ttaaacataa gatatattta taaaagagta tcaaagaatg caatacttac 3421 atctccactt ggttattaac gagtcgatgt ccatgagcag ggtgagcaac tggtgtggtt 3481 ggctgaacct gggttcatcc ctatagaagg tgatcatgat ggctccctga agggcacgat 3541 ggctaaaccg gcgatcccca cgacgcacca gtgcatcgtg cactgccgga tcaaagatgg 3601 agcgatacac ctcgcgtcgc ttctcaatgt ccatgaggcg gtagtttttc gccttctcca 3661 cgggctcctc catggcgctc tgtacctgcg cctccaggaa tcgatcgacg ctctcctgaa 3721 acttggccca gaagttgaag ccactctcct ccagtccggg cgtcctctcc agccatcgct 3781 gcactagctc cagtagcgag ggatctttct ccgagttgcg aatcgagttc cgcgcctcct 3841 cgtcgctaaa gacatccgag tacttctggt tgtatctcac ccgctgctct gtcagaactc 3901 ccagcttgtt ctcgatcaaa cggaactgca gcgactgaaa accagatgcg ggtgccaggt 3961 acttgcggaa gtccatgaag tctagcgggg tcatggtctc cagaatgggc acttggtcca 4021 ccaggagctg tacaaaggaa gttataaacg gattttggta agagattcag aaagcactca 4081 cttttagaat cagaaccact cggttcagtc gcttgacaat ctccagcgtc ttggtttcat 4141 cgatgacctc tgcatccaac atgtctcgta tggagtcgaa ctcaaagatg atctgcttga 4201 accaaagctc gtaggctgtg gcgaaggtac ttaaatgcca ttgagtgttg tcatcaaagt 4261 tgtaaaccta ctcaccctgg tgcgtgatga tgaacagatg ctcatcgtgc acgggtcgct 4321 tgtcctcctc ggacagcata cactgggcat ccagcagttt gtccagcatc agatactctc 4381 catagatttt gcccacttcc gtggttaatg gcaccgccga atcatcgtga tcgtttctgt 4441 atgggtttga attgaatcgc agaactgaag atcgattggc attcctggac agcacgtgct 4501 ggtgctcacc cgtttcctgc atagggacag ctcatggtgc acagctcaga tcagatcgtg 4561 actcctcgag cggcggatgc tggcgaactg atctccgcca gcggaccgga gatgagaccc 4621 cagcgaaccg ataacagagc gagagagctc cagttccgac tgattgcaca gtcggtgatc 4681 tgggcgatgg gcactgccag ataggctggg aattatcaat cacttgaggt gaaagtgcgg 4741 cgcacacaaa tccaagcttg atatcatcga tctcgaggct gcatccaacg cgttgggagc 4801 tctccggatc aattcggctt caggtaccgt cgacgatgta ggtcacggtc tcgaagccgc 4861 ggtgcgggtg ccagggcgtg cccttgggct ccccgggcgc gtactccacc tcacccatct 4921 ggtccatcat gatgaacggg tcgaggtggc ggtagttgat cccggcgaac gcgcggcgca 4981 ccgggaagcc ctcgccctcg aaaccgctgg gcgcggtggt cacggtgagc acgggacgtg 5041 cgacggcgtc ggctggtgcg gatacgcggg gcagcgtcag cgggttctcg acggtcacgg 5101 cgggcatgtc gacaagccga attgatccac tagaaggcct aattcggtac actagttggc 5161 cacgtaataa gtgtgcgttg aatttattcg caaaaacatt gcatattttc ggcaaagtaa 5221 aattttgttg cataccttat caaaaaataa gtgctgcata ctttttagag aaaccaaata 5281 attttttatt gcatacccgt ttttaataaa atacattgca taccctcttt taataaaaaa 5341 tattgcatac tttgacgaaa caaattttcg ttgcataccc aataaaagat tattatattg 5401 catacccgtt tttaataaaa tacattgcat accctctttt aataaagaat attgcatacg 5461 ttgacgaaac aaattttcgt tgcataccca ataaaagatt attatattgc ataccttttc 5521 ttgccatacc atttagccga tcaattgaga tctgaattct tttttgctca cctgtgattg 5581 ctcctactca aatacaaaaa catcaaattt tctgtcaata aagcatattt atttatattt 5641 attttacagg aaagaattcc ttttaaagtg tattttaacc tataatgaaa aacgattaaa 5701 aaaaatacat aaaataattc gaaaattttt gaatagccca ggttgataaa aattcatttc 5761 atacgtttta taacttatgc ccctaagtat tttttgacca tagtgtttca attctacatt 5821 aattttacag agtagaatga aacgccacct actcagccaa gaggcgaaaa ggttagctcg 5881 ccaagcagag agggcgccag tgctcactac tttttataat tctcaacttc tttttccaga 5941 ctcagttcgt atatatagac ctattttcaa tttaacgtcg gggtcttcga gaagacctgt 6001 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg 6061 caccgagtcg gtgctttttt gcctacctgg agcctgagag ttgttcaata aaataaaaat 6121 gtttcgtttt tttgctttcg ccagtattta ttatttttca tcaatatgta ttcaatttgg 6181 tatgtattta gtaattgtaa tatatagaca atggttttcc gttgacgtac atacatctga 6241 cgtgtgttta tttagacata atagttatgt tttcacatct ttttaatgtt cgcttaatgc 6301 gtatgcattc tagacaattg tgctcggcaa cagtatattt gtggtgtgcc aaccaacaac 6361 ctgcagga //