LOCUS Exported 7791 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Ubiquitous expression of one or multiple sgRNAs in Drosophila. Selection marker: Mini-white.. ACCESSION . VERSION . KEYWORDS pCFD5_w SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7791) TITLE crisprflydesign.org JOURNAL Unpublished REFERENCE 2 (bases 1 to 7791) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7791 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 54..123 /label=attB /bound_moiety="phage phi-C31 integrase" /note="attB site for the phi-C31 integrase (Groth et al., 2000)" promoter complement(342..360) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(381..397) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(381..397) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(394..416) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 405..421 /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(429..459) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 474..495 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(612..629) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(783..1371) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(863..882) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(1542..2402) /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 2165..2184 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(2403..2507) /gene="bla" /label=AmpR promoter rep_origin complement(2533..2988) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2670..2691) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 2882..2901 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3115..3137 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 3129..3146 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 3130..3146 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 3153..3172 /label=T7 /note="T7 promoter, forward primer" promoter 3153..3171 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(3205..3223) /label=pCasper-F /note="Drosophila mini-white gene, reverse primer" CDS join(3675..3746,4148..4421,4496..5150,5212..5527,5748..5879, 5950..6564) /codon_start=1 /gene="white" /product="Drosophila white gene eye color pigment" /label=mini-white /note="This modified version of the white gene lacks part of the first intron." /translation="MGQEDQELLIRGGSKHPSAEHLNNGDSGAASQSCINQGFGQAKNY GTLRPPSPPEDSGSGSGQLAENLTYAWHNMDIFGAVNQPGSGWRQLVNRTRGLFCNERH IPAPRKHLLKNVCGVAYPGELLAVMGSSGAGKTTLLNALAFRSPQGIQVSPSGMRLLNG QPVDAKEMQARCAYVQQDDLFIGSLTAREHLIFQAMVRMPRHLTYRQRVARVDQVIQEL SLSKCQHTIIGVPGRVKGLSGGERKRLAFASEALTDPPLLICDEPTSGLDSFTAHSVVQ VLKKLSQKGKTVILTIHQPSSELFELFDKILLMAEGRVAFLGTPSEAVDFFSYVGAQCP TNYNPADFYVQVLAVVPGREIESRDRIAKICDNFAISKVARDMEQLLATKNLEKPLEQP ENGYTYKATWFMQFRAVLWRSWLSVLKEPLLVKVRLIQTTMVAILIGLIFLGQQLTQVG VMNINGAIFLFLTNMTFQNVFATINVFTSELPVFMREARSRLYRCDTYFLGKTIAELPL FLTVPLVFTAIAYPMIGLRAGVLHFFNCLALVTLVANVSTSFGYLISCASSSTSMALSV GPPVIIPFLLFGGFFLNSGSVPVYLKWLSYLSWFRYANEGLLINQWADVEPGEISCTSS NTTCPSSGKVILETLNFSAADLPLDYVGLAILIVSFRVLAYLALRLRARRKE" promoter 6788..7208 /label=dU6-3 promoter /note="RNA polymerase III promoter for Drosophila U6-3 snRNA (Port et al., 2014)" misc_RNA 7314..7389 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" misc_RNA 7476..7551 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" ORIGIN 1 gtggtgtgcc aaccaacaac ggatccacta gtgtcgacga tgtaggtcac agtctcgaag 61 ccgcggtgcg ggtgccaggg cgtgcccttg ggctccccgg gcgcgtactc cacctcaccc 121 atctggtcca tcatgatgaa cgggtcgagg tggcggtagt tgatcccggc gaacgcgcgg 181 cgcaccggga agccctcgcc ctcgaaaccg ctgggcgcgg tggtcacggt gagcacggga 241 cgtgcgacgg cgtcggcggg tgcggatacg cggggcagcg tcagcgggtt ctcgacggtc 301 acggcgggca tgtcgacact agttctagcc agcttttgtt ccctttagtg agggttaatt 361 tcgagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca 421 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg 481 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg 541 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 601 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 661 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 721 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 781 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 841 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 901 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 961 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 1021 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 1081 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 1141 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 1201 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 1261 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 1321 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 1381 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 1441 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 1501 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 1561 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 1621 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 1681 cgggacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 1741 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 1801 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 1861 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 1921 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 1981 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 2041 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 2101 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 2161 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 2221 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 2281 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 2341 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 2401 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 2461 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 2521 aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt 2581 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag 2641 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga 2701 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg 2761 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc 2821 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg 2881 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc 2941 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat 3001 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc 3061 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt 3121 cacgacgttg taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg 3181 gtacgtaccg ggcccctagt atgtatgtaa gttaataaaa cccatttttg cggaaagtag 3241 ataaaaaaaa catttttttt ttttactgca ctggatatca ttgaacttat ctgatcagtt 3301 ttaaatttac ttcgatccaa gggtatttga tgtaccaggt tctttcgatt acctctcact 3361 caaaatgaca ttccactcaa agtcagcgct gtttgcctcc ttctctgtcc acagaaatat 3421 cgccgtctct ttcgccgctg cgtccgctat ctctttcgcc accgtttgta gcgttacgta 3481 gcgtcaatgt ccgccttcag ttgcattttg tcagcggttt cgtgacgaag ctccaagcgg 3541 tttacgccat caattaaaca caaagtgctg tgccaaaact cctctcgctt cttatttttg 3601 tttgtttttt gagtgattgg ggtggtgatt ggttttgggt gggtaagcag gggaaagtgt 3661 gaaaaatccc ggcaatgggc caagaggatc aggagctatt aattcgcgga ggcagcaaac 3721 acccatctgc cgagcatctg aacaatgtga gtagtacatg tgcatacatc ttaagttcac 3781 ttgatctata ggaactgcga ttgcaacatc aaattgtctg cggcgtgaga actgcgaccc 3841 acaaaaatcc caaaccgcaa ttgcacaaac aaatagtgac acgaaacaga ttattctggt 3901 agctgttctc gctatataag acaatttttg agatcatatc atgatcaaga catctaaagg 3961 cattcatttt cgactatatt cttttttaca aaaaatataa caaccagata ttttaagctg 4021 atcctagatg cacaaaaaat aaataaaagt ataaacctac ttcgtaggat acttcggggt 4081 actttttgtt cggggttaga tgagcataac gcttgtagtt gatatttgag atcccctatc 4141 attgcagggt gacagcggag cggcttcgca gagctgcatt aaccagggct tcgggcaggc 4201 caaaaactac ggcacgctcc ggccacccag tccgccggag gactccggtt cagggagcgg 4261 ccaactagcc gagaacctca cctatgcctg gcacaatatg gacatctttg gggcggtcaa 4321 tcagccgggc tccggatggc ggcagctggt caaccggaca cgcggactat tctgcaacga 4381 gcgacacata ccggcgccca ggaaacattt gctcaagaac ggtgagtttc tattcgcagt 4441 cggctgatct gtgtgaaatc ttaataaagg gtccaattac caatttgaaa ctcagtttgc 4501 ggcgtggcct atccgggcga acttttggcc gtgatgggca gttccggtgc cggaaagacg 4561 accctgctga atgcccttgc ctttcgatcg ccgcagggca tccaagtatc gccatccggg 4621 atgcgactgc tcaatggcca acctgtggac gccaaggaga tgcaggccag gtgcgcctat 4681 gtccagcagg atgacctctt tatcggctcc ctaacggcca gggaacacct gattttccag 4741 gccatggtgc ggatgccacg acatctgacc tatcggcagc gagtggcccg cgtggatcag 4801 gtgatccagg agctttcgct cagcaaatgt cagcacacga tcatcggtgt gcccggcagg 4861 gtgaaaggtc tgtccggcgg agaaaggaag cgtctggcat tcgcctccga ggcactaacc 4921 gatccgccgc ttctgatctg cgatgagccc acctccggac tggactcatt taccgcccac 4981 agcgtcgtcc aggtgctgaa gaagctgtcg cagaagggca agaccgtcat cctgaccatt 5041 catcagccgt catccgagct gtttgagctc tttgacaaga tccttctgat ggccgagggc 5101 agggtagctt tcttgggcac tcccagcgaa gccgtcgact tcttttccta gtgagttcga 5161 tgtgtttatt aagggtatct agcattacat tacatctcaa ctcctatcca gcgtgggtgc 5221 ccagtgtcct accaactaca atccggcgga cttttacgta caggtgttgg ccgttgtgcc 5281 cggacgggag atcgagtccc gtgatcggat cgccaagata tgcgacaatt ttgctattag 5341 caaagtagcc cgggatatgg agcagttgtt ggccaccaaa aatttggaga agccactgga 5401 gcagccggag aatgggtaca cctacaaggc cacctggttc atgcagttcc gggcggtcct 5461 gtggcgatcc tggctgtcgg tgctcaagga accactcctc gtaaaagtgc gacttattca 5521 gacaacggtg agtggttcca gtggaaacaa atgatataac gcttacaatt cttggaaaca 5581 aattcgctag attttagtta gaattgcctg attccacacc cttcttagtt tttttcaatg 5641 agatgtatag tttatagttt tgcagaaaat aaataaattt catttaactc gcgaacatgt 5701 tgaagatatg aatattaatg agatgcgagt aacattttaa tttgcagatg gttgccatct 5761 tgattggcct catctttttg ggccaacaac tcacgcaagt gggcgtgatg aatatcaacg 5821 gagccatctt cctcttcctg accaacatga cctttcaaaa cgtctttgcc acgataaatg 5881 taagtcttgt ttagaataca tttgcatatt aataatttac taactttcta atgaatcgat 5941 tcgatttagg tgttcacctc agagctgcca gtttttatga gggaggcccg aagtcgactt 6001 tatcgctgtg acacatactt tctgggcaaa acgattgccg aattaccgct ttttctcaca 6061 gtgccactgg ttttcacggc gattgcctat ccgatgatcg gactgcgggc cggagtgctg 6121 cacttcttca actgcctggc gctggtcact ctggtggcca atgtgtcaac gtccttcgga 6181 tatctaatat cctgcgccag ctcctcgacc tcgatggcgc tgtctgtggg tccgccggtt 6241 atcataccat tcctgctctt tggcggcttc ttcttgaact cgggctcggt gccagtatac 6301 ctcaaatggt tgtcgtacct ctcatggttc cgttacgcca acgagggtct gctgattaac 6361 caatgggcgg acgtggagcc gggcgaaatt agctgcacat cgtcgaacac cacgtgcccc 6421 agttcgggca aggtcatcct ggagacgctt aacttctccg ccgccgatct gccgctggac 6481 tacgtgggtc tggccattct catcgtgagc ttccgggtgc tcgcatatct ggctctaaga 6541 cttcgggccc gacgcaagga gtagccgaca tatatccgaa ataactgctt gttttttttt 6601 tttaccatta ttaccatcgt gtttactgtt tattgccccc tcaaaaagct aatgtaatta 6661 tatttgtgcc aataaaaaca agatatgacc tatagaatac aagtatttcc ccttcgaaca 6721 tccccacaag tagactttgg atttgtttct aaccaaaaga cttacacacc tgcatacctt 6781 agaattcttt tttgctcacc tgtgattgct cctactcaaa tacaaaaaca tcaaattttc 6841 tgtcaataaa gcatatttat ttatatttat tttacaggaa agaattcctt ttaaagtgta 6901 ttttaaccta taatgaaaaa cgattaaaaa aaatacataa aataattcga aaatttttga 6961 atagcccagg ttgataaaaa ttcatttcat acgttttata acttatgccc ctaagtattt 7021 tttgaccata gtgtttcaat tctacattaa ttttacagag tagaatgaaa cgccacctac 7081 tcagccaaga ggcgaaaagg ttagctcgcc aagcagagag ggcgccagtg ctcactactt 7141 tttataattc tcaacttctt tttccagact cagttcgtat atatagacct attttcaatt 7201 taacgtcggg gctttgagtg tgtgtagaca tcaagcatcg gtggttcagt ggtagaatgc 7261 tcgcctgcca cgcgggcggc ccgggttcga ttcccggccg atgcagggtc ttcgttttag 7321 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg 7381 agtcggtgct aacaaagcac cagtggtcta gtggtagaat agtaccctgc cacggtacag 7441 acccgggttc gattcccggc tggtgcagaa gacctgtttt agagctagaa atagcaagtt 7501 aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttgcc 7561 tacctggagc ctgagagttg ttcaataaaa taaaaatgtt tcgttttttt gctttcgcca 7621 gtatttatta tttttcatca atatgtattc aatttggtat gtatttagta attgtaatat 7681 atagacaatg gttttccgtt gacgtacata catctgacgt gtgtttattt agacataata 7741 gttatgtttt cacatctttt taatgttcgc ttaatgcgta tgcattctag a //