LOCUS Exported 8853 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expresses gRNAs targeting hid and eve. ACCESSION . VERSION . KEYWORDS OA-1045A SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8853) AUTHORS . TITLE Engineering reproductive isolation in D. melanogaster JOURNAL Unpublished REFERENCE 2 (bases 1 to 8853) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8853 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 57..79 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 71..88 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 72..88 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 95..114 /label=T7 /note="T7 promoter, forward primer" promoter 95..113 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(147..165) /label=pCasper-F /note="Drosophila mini-white gene, reverse primer" CDS join(617..688,1090..1363,1438..2092,2154..2469,2673..2804, 2875..3489) /codon_start=1 /gene="white" /product="Drosophila white gene eye color pigment" /label=mini-white /note="This is a modified version of the white gene lacking part of the first intron." /translation="MGQEDQELLIRGGSKHPSAEHLNNGDSGAASQSCINQGFGQAKNY GTLRPPSPPEDSGSGSGQLAENLTYAWHNMDIFGAVNQPGSGWRQLVNRTRGLFCNERH IPAPRKHLLKNVCGVAYPGELLAVMGSSGAGKTTLLNALAFRSPQGIQVSPSGMRLLNG QPVDAKEMQARCAYVQQDDLFIGSLTAREHLIFQAMVRMPRHLTYRQRVARVDQVIQEL SLSKCQHTIIGVPGRVKGLSGGERKRLAFASEALTDPPLLICDEPTSGLDSFTAHSVVQ VLKKLSQKGKTVILTIHQPSSELFELFDKILLMAEGRVAFLGTPSEAVDFFSYVGAQCP TNYNPADFYVQVLAVVPGREIESRDRIAKICDNFAISKVARDMEQLLATKNLEKPLEQP ENGYTYKATWFMQFRAVLWRSWLSVLKEPLLVKVRLIQTTMVAILIGLIFLGQQLTQVG VMNINGAIFLFLTNMTFQNVFATINVFTSELPVFMREARSRLYRCDTYFLGKTIAELPL FLTVPLVFTAIAYPMIGLRAGVLHFFNCLALVTLVANVSTSFGYLISCASSSTSMALSV GPPVIIPFLLFGGFFLNSGSVPVYLKWLSYLSWFRYANEGLLINQWADVEPGEISCTSS NTTCPSSGKVILETLNFSAADLPLDYVGLAILIVSFRVLAYLALRLRARRKE" protein_bind complement(4264..4297) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 4356..4776 /label=dU6-3 promoter /note="RNA polymerase III promoter for Drosophila U6-3 snRNA (Port et al., 2014)" misc_RNA 4796..4871 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 4886..5682 /label=dU6-1 promoter /note="RNA polymerase III promoter for Drosophila U6-1 snRNA (Port et al., 2014)" misc_RNA 5701..5776 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" protein_bind 5849..5918 /label=attB /bound_moiety="phage phi-C31 integrase" /note="attB site for the phi-C31 integrase (Groth et al., 2000)" promoter complement(6137..6155) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(6176..6192) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6176..6192) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(6189..6211) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 6200..6216 /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(6224..6254) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6269..6290 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6407..6424) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6578..7166) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6658..6677) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7337..8197) /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 7960..7979 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8198..8302) /gene="bla" /label=AmpR promoter rep_origin complement(8328..8783) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(8465..8486) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 8677..8696 /label=F1ori-R /note="F1 origin, reverse primer" ORIGIN 1 gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca 61 gtcacgacgt tgtaaaacga cggccagtga attgtaatac gactcactat agggcgaatt 121 gggtacgtac cgggccccta gtatgtatgt aagttaataa aacccatttt tgcggaaagt 181 agataaaaaa aacatttttt ttttttactg cactggatat cattgaactt atctgatcag 241 ttttaaattt acttcgatcc aagggtattt gatgtaccag gttctttcga ttacctctca 301 ctcaaaatga cattccactc aaagtcagcg ctgtttgcct ccttctctgt ccacagaaat 361 atcgccgtct ctttcgccgc tgcgtccgct atctctttcg ccaccgtttg tagcgttacg 421 tagcgtcaat gtccgccttc agttgcattt tgtcagcggt ttcgtgacga agctccaagc 481 ggtttacgcc atcaattaaa cacaaagtgc tgtgccaaaa ctcctctcgc ttcttatttt 541 tgtttgtttt ttgagtgatt ggggtggtga ttggttttgg gtgggtaagc aggggaaagt 601 gtgaaaaatc ccggcaatgg gccaagagga tcaggagcta ttaattcgcg gaggcagcaa 661 acacccatct gccgagcatc tgaacaatgt gagtagtaca tgtgcataca tcttaagttc 721 acttgatcta taggaactgc gattgcaaca tcaaattgtc tgcggcgtga gaactgcgac 781 ccacaaaaat cccaaaccgc aattgcacaa acaaatagtg acacgaaaca gattattctg 841 gtagctgttc tcgctatata agacaatttt tgagatcata tcatgatcaa gacatctaaa 901 ggcattcatt ttcgactata ttctttttta caaaaaatat aacaaccaga tattttaagc 961 tgatcctaga tgcacaaaaa ataaataaaa gtataaacct acttcgtagg atacttcggg 1021 gtactttttg ttcggggtta gatgagcata acgcttgtag ttgatatttg agatccccta 1081 tcattgcagg gtgacagcgg agcggcttcg cagagctgca ttaaccaggg cttcgggcag 1141 gccaaaaact acggcacgct ccggccaccc agtccgccgg aggactccgg ttcagggagc 1201 ggccaactag ccgagaacct cacctatgcc tggcacaata tggacatctt tggggcggtc 1261 aatcagccgg gctccggatg gcggcagctg gtcaaccgga cacgcggact attctgcaac 1321 gagcgacaca taccggcgcc caggaaacat ttgctcaaga acggtgagtt tctattcgca 1381 gtcggctgat ctgtgtgaaa tcttaataaa gggtccaatt accaatttga aactcagttt 1441 gcggcgtggc ctatccgggc gaacttttgg ccgtgatggg cagttccggt gccggaaaga 1501 cgaccctgct gaatgccctt gcctttcgat cgccgcaggg catccaagta tcgccatccg 1561 ggatgcgact gctcaatggc caacctgtgg acgccaagga gatgcaggcc aggtgcgcct 1621 atgtccagca ggatgacctc tttatcggct ccctaacggc cagggaacac ctgattttcc 1681 aagccatggt gcggatgcca cgacatctga cctatcggca gcgagtggcc cgcgtggatc 1741 aggtgatcca ggagctttcg ctcagcaaat gtcagcacac gatcatcggt gtgcccggca 1801 gggtgaaagg tctgtccggc ggagaaagga agcgtctggc attcgcctcc gaggctctaa 1861 ccgatccgcc gcttctgatc tgcgatgagc ccacctccgg actggactcc tttaccgccc 1921 acagcgtcgt ccaggtgctg aagaagctgt cgcagaaggg caagaccgtc atcctgacca 1981 ttcatcagcc gtcttccgag ctgtttgagc tctttgacaa gatccttctg atggccgagg 2041 gcagggtagc tttcttgggc actcccagcg aagccgtcga cttcttttcc tagtgagttc 2101 gatgtgttta ttaagggtat ctagtattac ataacatctc aactcctatc cagcgtgggt 2161 gcccagtgtc ctaccaacta caatccggcg gacttttacg tacaggtgtt ggccgttgtg 2221 cccggacggg agatcgagtc ccgtgatcgg atcgccaaga tatgcgacaa ttttgccatt 2281 agcaaagtag cccgggatat ggagcagttg ttggccacca aaaatctgga gaagccactg 2341 gagcagccgg agaatgggta cacctacaag gccacctggt tcatgcagtt ccgggcggtc 2401 ctgtggcgat cctggctgtc ggtgctcaag gaaccactcc tcgtaaaagt gcgacttatt 2461 cagacaacgg tgagtggttc cagtggaaac aaatgatata acgcttacaa ttcttggaaa 2521 caaattcgct agattttaga tagaattgcc tgattccaca cccttcttag tttttttcaa 2581 tgagatgtat agtttatagt tttgcagaag ataaataaat ttcatttaac tcgcgaatat 2641 taatgagatg cgagtaacat tttaatttgc agatggttgc catcttgatt ggcctcatct 2701 ttttgggcca acaactcacg caagtgggtg tgatgaatat caacggagcc atcttcctct 2761 tcctgaccaa catgaccttt caaaacgtct ttgccacgat aaatgtaagt catgtttaga 2821 atacatttgc atttcaataa tttactaact ttctaatgaa tcgattcgat ttaggtgttc 2881 acctcagagc tgccagtttt tatgagggag gcccgaagtc gactttatcg ctgtgacaca 2941 tactttctgg gcaaaacgat tgccgaattg ccgctttttc tcacagtgcc actggtcttc 3001 acggcgattg cctatccgat gatcggactg cgggccggag tgctgcactt cttcaactgc 3061 ctggcgctgg tcactctggt ggccaatgtg tcaacgtcct tcggatatct aatatcctgc 3121 gccagctcct cgacctcgat ggcgctgtct gtgggtccgc cggttatcat accattcctg 3181 ctctttggcg gcttcttctt gaactcgggc tcggtgccag tatacctcaa atggttgtcg 3241 tacctctcat ggttccgtta cgccaacgag ggtctgctga ttaaccaatg ggcggacgtg 3301 gagccgggcg aaattagctg cacatcgtcg aacaccacgt gccccagttc gggcaaggtc 3361 atcctggaga cgcttaactt ctccgccgcc gatctgccgc tggactacgt gggtctggcc 3421 attctcatcg tgagcttccg ggtgctcgca tatctggctc taagacttcg ggcccgacgc 3481 aaggagtagc cgacatatat ccgaaataac tgcttgtttt ttttttttac cattattacc 3541 atcgtgttta ctgtttattg ccccctcaaa aagctaatgt aattatattt gtgccaataa 3601 aaacaagata tgacctatag aatacaagta tttccccttc gaacatcccc acaagtagac 3661 tttggatttg tcttctaacc aaaagactta cacacctgca taccttacat caaaaactcg 3721 tttatcgcta cataaaacac cgggatatat tttttatata catacttttc aaatcgcgcg 3781 ccctcttcat aattcacctc caccacacca cgtttcgtag ttgctctttc gctgtctccc 3841 acccgctctc cgcaacacat tcaccttttg ttcgacgacc ttggagcgac tgtcgttagt 3901 tccgcgcgat tcggttcgct caaatggttc cgagtggttc atttcgtctc aatagaaatt 3961 agtaataaat atttgtatgt acaatttatt tgctccaata tatttgtata tatttccctc 4021 acagctatat ttattctaat ttaatattat gactttttaa ggtaattttt tgtgacctgt 4081 tcggagtgat tagcgttaca atttgaactg aaagtgacat ccagtgtttg ttccttgtgt 4141 agatgcatct caaaaaaatg gtgggcataa tagtgttgtt tatatatatc aaaaataaca 4201 actataataa taagaataca tttaatttag aaaatgcttg gatttcactg gaactaggct 4261 agcataactt cgtataatgt atgctatacg aagttatgct agcggatccg ggaattggga 4321 attgggcaat atttaaatgg cggcgcgccg aattcttttt tgctcacctg tgattgctcc 4381 tactcaaata caaaaacatc aaattttctg tcaataaagc atatttattt atatttattt 4441 tacaggaaag aattcctttt aaagtgtatt ttaacctata atgaaaaacg attaaaaaaa 4501 atacataaaa taattcgaaa atttttgaat agcccaggtt gataaaaatt catttcatac 4561 gttttataac ttatgcccct aagtattttt tgaccatagt gtttcaattc tacattaatt 4621 ttacagagta gaatgaaacg ccacctactc agccaagagg cgaaaaggtt agctcgccaa 4681 gcagagaggg cgccagtgct cactactttt tataattctc aacttctttt tccagactca 4741 gttcgtatat atagacctat tttcaattta acgtcggatc gtgcggtgct gagaggtttt 4801 agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac 4861 cgagtcggtg cttttttgtt taaacatttt caacgtcctc gatagtatag tggttagtat 4921 ccccgcctgt cacgcgggag accggggttc aattccccgt cggggagaat ctgtgattct 4981 tttttttttt cttttacttt gttatataaa caatttttgt tttaattgaa tctaatttgc 5041 cattgctttt aggaatctca ggcatccagc aagcgtttgt ccgccgaatc gcccatcagt 5101 gaagaagatc ctgtggcggc tacgaaaatc tccccggcca tgtcggcctc cacctccagc 5161 gaaaaaccca tcagcgagct ggccacctct gtgctgaccc accgctttcc agactccacc 5221 tcctcacccg gcgaacatgg ccttggacga atgcagttgt cgatccgcta cagcgcccag 5281 cgtcaaaaac tagacgtgac catacacaaa atccagaaga taccacttcg cgatcccagc 5341 aatatccccg atccgtatgt taagctgtat ctgttgcctg gacgcaccaa ggagtcgaaa 5401 cgcaagacga gcgtgatcaa ggacaactgc aaccccgtct acgatgcatc ctttgagtac 5461 ctgatttcca ttgccgaact caggcagacg gaactggagg tgacggtgtg cacccaaaag 5521 ggattcctat ccggcggtag tcccatcatt ggcatggtag gtacccgaaa gcaacccctt 5581 agttacagac acagcgcgta cgtccttcgc atccttatga ttcccaagta catattctgc 5641 aagagtacag tatatatagg aaagatatcc gggtgaactt cggcacatgc acgtgcatga 5701 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 5761 ggcaccgagt cggtgctttt ttaattaagg aacgcggccg caacggtacc tctagagatc 5821 cactagtgtc gacgatgtag gtcacggtct cgaagccgcg gtgcgggtgc cagggcgtgc 5881 ccttgggctc cccgggcgcg tactccacct cacccatctg gtccatcatg atgaacgggt 5941 cgaggtggcg gtagttgatc ccggcgaacg cgcggcgcac cgggaagccc tcgccctcga 6001 aaccgctggg cgcggtggtc acggtgagca cgggacgtgc gacggcgtcg gcgggtgcgg 6061 atacgcgggg cagcgtcagc gggttctcga cggtcacggc gggcatgtcg acactagttc 6121 tagccagctt ttgttccctt tagtgagggt taatttcgag cttggcgtaa tcatggtcat 6181 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa 6241 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc 6301 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc 6361 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact 6421 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 6481 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 6541 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 6601 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 6661 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 6721 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac 6781 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 6841 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 6901 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 6961 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa 7021 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 7081 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 7141 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 7201 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 7261 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 7321 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 7381 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 7441 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 7501 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 7561 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 7621 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 7681 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 7741 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 7801 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 7861 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 7921 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca 7981 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 8041 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 8101 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa 8161 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt 8221 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 8281 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt 8341 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata 8401 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt 8461 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg 8521 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt 8581 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc 8641 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg 8701 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct 8761 taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg 8821 gcgatcggtg cgggcctctt cgctattacg cca //