LOCUS Exported 8132 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION to create unc-73E null allele. ACCESSION . VERSION . KEYWORDS TU#1806_CRISPR_unc-73_exon21 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8132) AUTHORS Zheng C, Diaz-Cuadros M, Chalfie M TITLE GEFs and Rac GTPases control directional specificity of neurite extension along the anterior-posterior axis. JOURNAL Proc Natl Acad Sci U S A. 2016 Jun 21;113(25):6973-8. doi: 10.1073/pnas.1607179113. Epub 2016 Jun 6. PUBMED 27274054 REFERENCE 2 (bases 1 to 8132) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8132 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(21..40) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 834..1422 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1323..1342 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 1549..1571 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 1568..1584 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 1568..1584 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" protein_bind 1604..1624 /gene="mutant version of attB" /label=attB4 /bound_moiety="BP Clonase(TM)" /note="core recombination site for the Gateway(R) BP reaction" promoter 1625..2224 /gene="eft-3" /label=eft-3 promoter /note="promoter for the C. elegans elongation factor 1-alpha gene (Mitrovich and Anderson, 2000)" CDS join(2241..3284,3336..4406,4458..5483,5535..6497) /codon_start=1 /product="S. pyogenes Cas9, codon optimized for C. elegans with synthetic introns" /label=Cas9 /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 6513..6533 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 6534..6560 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" terminator 6575..6898 /gene="tbb-2" /label=tbb-2 terminator /note="transcription terminator for the C. elegans tubulin beta-2 gene" protein_bind complement(6902..6922) /gene="mutant version of attB" /label=attB3 /bound_moiety="BP Clonase(TM)" /note="core recombination site for the Gateway(R) BP reaction" primer_bind complement(6930..6947) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(6930..6946) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6939..6961) /label=M13/pUC Forward /note="In lacZ gene" promoter 6967..7316 /label=CeU6 promoter /note="promoter for the C. elegans R07E5.16 U6 snRNA gene (Dickinson et al., 2013)" misc_RNA 7337..7412 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" primer_bind 7684..7706 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(7744..7762) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 7830..7934 /gene="bla" /label=AmpR promoter CDS 7935..663 /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" ORIGIN 1 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa 61 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc 121 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc 181 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta 241 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag 301 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca 361 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata 421 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc 481 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca 541 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca 601 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg 661 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 721 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 781 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 841 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 901 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 961 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac 1021 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 1081 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 1141 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 1201 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag 1261 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 1321 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 1381 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 1441 tttttacggt tcctggtctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc 1501 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccag cggataacaa 1561 tttcacacag gaaacagcta tgaccatgat tacgccaagc tatcaacttt gtatagaaaa 1621 gttggcacct ttggtctttt attgtcaact tccattggtt cttccattgt ttctgttaaa 1681 ttaatgaatt tttcataaaa taaagacatt atacaatata aaaatgaaga atttattgaa 1741 aataaactgc cagagagaaa aagtatgcaa cactcccgcc gagagtgttt gaaatggtgt 1801 acggtacatt ttcgtgctag gagttagatg tgcaggcagc aacgagaggg ggagagattt 1861 ttttgggcct tgtgaaatta acgtgagttt tctggtcatc tgactaatca tgttggtttt 1921 ttgttggttt attttgtttt tatctttgtt tttatccaga ttaggaaatt taaattttat 1981 gaatttataa tgaggtcaaa cattcagtcc cagcgttttt cctgttctca ctgtttagtc 2041 gaatttttat tttaggcttt caacaaatgt tctaactgtc ttatttgtga cctcactttt 2101 tatatttttt taatttttaa aaatattaga agtttctagg ataatttttt cgacttttat 2161 tctctctacc gtccgcactc ttcttacttt taaattaaat tgtttttttt tcagttggga 2221 aacactttgc tctagaaaaa atggacaaaa aatacagcat cggccttgac atcggaacga 2281 actccgtcgg atgggccgtc atcaccgacg agtacaaggt cccatccaag aagttcaagg 2341 tcctcggaaa caccgaccgt cactccatca agaagaacct catcggagcc ctcctcttcg 2401 actccggaga gaccgccgag gccacccgtc tcaagcgtac cgcccgtcgt cgttacaccc 2461 gtcgtaagaa ccgtatctgc tacctccaag agatcttctc caacgagatg gccaaggtcg 2521 acgactcctt cttccaccgt ctcgaggagt ccttcctcgt cgaggaggac aagaagcacg 2581 agcgtcaccc aatcttcgga aacatcgtcg acgaggtcgc ctaccacgag aagtacccaa 2641 ccatctacca cctccgtaag aagctcgtcg actccaccga caaggccgac ctccgtctca 2701 tctacctcgc cctcgcccac atgatcaagt tccgtggaca cttcctcatc gagggagacc 2761 tcaacccaga caactccgac gtcgacaagc tcttcatcca actcgtccaa acctacaacc 2821 aactcttcga ggagaaccca atcaacgcct ccggagtcga cgccaaggcc atcctctccg 2881 cccgtctctc caagtcccgt cgtctcgaga acctcatcgc ccaactccca ggagagaaga 2941 agaacggact cttcggaaac ctcatcgccc tctccctcgg actcacccca aacttcaagt 3001 ccaacttcga cctcgccgag gacgccaagc tccaactctc caaggacacc tacgacgacg 3061 acctcgacaa cctcctcgcc caaatcggag accaatacgc cgacctcttc ctcgccgcca 3121 agaacctctc cgacgccatc ctcctctccg acatcctccg tgtcaacacc gagatcacca 3181 aggccccact ctccgcctcc atgatcaagc gttacgacga gcaccaccaa gacctcaccc 3241 tcctcaaggc cctcgtccgt caacaactcc cagagaagta caaggtaagt ttaaacatat 3301 atatactaac taaccctgat tatttaaatt ttcaggagat cttcttcgac caatccaaga 3361 acggatacgc cggatacatc gacggaggag cctcccaaga ggagttctac aagttcatca 3421 agccaatcct cgagaagatg gacggaaccg aggagctcct cgtcaagctc aaccgtgagg 3481 acctcctccg taagcaacgt accttcgaca acggatccat cccacaccaa atccacctcg 3541 gagagctcca cgccatcctc cgtcgtcaag aggacttcta cccattcctc aaggacaacc 3601 gtgagaagat cgagaagatc ctcaccttcc gtatcccata ctacgtcgga ccactcgccc 3661 gtggaaactc ccgtttcgcc tggatgaccc gtaagtccga ggagaccatc accccatgga 3721 acttcgagga ggtcgtcgac aagggagcct ccgcccaatc cttcatcgag cgtatgacca 3781 acttcgacaa gaacctccca aacgagaagg tcctcccaaa gcactccctc ctctacgagt 3841 acttcaccgt ctacaacgag ctcaccaagg tcaagtacgt caccgaggga atgcgtaagc 3901 cagccttcct ctccggagag caaaagaagg ccatcgtcga cctcctcttc aagaccaacc 3961 gtaaggtcac cgtcaagcaa ctcaaggagg actacttcaa gaagatcgag tgcttcgact 4021 ccgtcgagat ctccggagtc gaggaccgtt tcaacgcctc cctcggaacc taccacgacc 4081 tcctcaagat catcaaggac aaggacttcc tcgacaacga ggagaacgag gacatcctcg 4141 aggacatcgt cctcaccctc accctcttcg aggaccgtga gatgatcgag gagcgtctca 4201 agacctacgc ccacctcttc gacgacaagg tcatgaagca gcttaagcgt cgtcgttaca 4261 ccggatgggg acgtctctcc cgtaagctca tcaacggaat ccgtgacaag caatccggaa 4321 agaccatcct cgacttcctc aagtccgacg gattcgccaa ccgtaacttc atgcaactca 4381 tccacgacga ctccctcacc ttcaaggtaa gtttaaacag ttcggtacta actaaccata 4441 catatttaaa ttttcaggag gacatccaaa aggcccaagt ctccggacaa ggagactccc 4501 tccacgagca catcgccaac ctcgccggat ccccagccat caagaaggga atcctccaaa 4561 ccgtcaaggt cgtcgacgag ctcgtcaagg tcatgggacg tcacaagcca gagaacatcg 4621 tcatcgagat ggcccgtgag aaccaaacca cccaaaaggg acaaaagaac tcccgtgagc 4681 gtatgaagcg tatcgaggag ggaatcaagg agctcggatc ccaaatcctc aaggagcacc 4741 cagtcgagaa cacccaactc caaaacgaga agctctacct ctactacctc caaaacggac 4801 gtgacatgta cgtcgaccaa gagctcgaca tcaaccgtct ctccgactac gacgtcgacc 4861 acatcgtccc acaatccttc ctcaaggacg actccatcga caacaaggtc ctcacccgtt 4921 ccgacaagaa ccgtggaaag tccgacaacg tcccatccga ggaggtcgtc aagaagatga 4981 agaactactg gcgtcaactc ctcaacgcca agctcatcac ccaacgtaag ttcgacaacc 5041 tcaccaaggc tgagcgtgga ggactctccg agctcgacaa ggccggattc atcaagcgtc 5101 aactcgtcga gacccgtcaa atcaccaagc acgtcgccca aatcctcgac tcccgtatga 5161 acaccaagta cgacgagaac gacaagctca tccgtgaggt caaggtcatc accctcaagt 5221 ccaagctcgt ctccgacttc cgtaaggact tccaattcta caaggtccgt gagatcaaca 5281 actaccacca cgcccacgac gcctacctca acgccgtagt aggaaccgcc ctcatcaaga 5341 agtacccaaa gctcgagtcc gagttcgtct acggagacta caaggtctac gacgtccgta 5401 agatgatcgc caagtccgag caagagatcg gaaaggccac cgccaagtac ttcttctact 5461 ccaacatcat gaacttcttc aaggtaagtt taaacatgat tttactaact aactaatctg 5521 atttaaattt tcagaccgag atcaccctcg ccaacggaga gatccgtaag cgtccactca 5581 tcgagaccaa cggagagacc ggagagatcg tctgggacaa gggacgtgac ttcgccacag 5641 tccgtaaggt cctctccatg ccacaagtca acatcgtcaa gaagaccgag gtccaaaccg 5701 gaggattctc caaggagtcc atcctcccaa agcgtaactc cgacaagctc atcgcccgta 5761 agaaggactg ggacccaaag aagtacggag gattcgactc cccaaccgtc gcctactccg 5821 tcctcgtcgt cgccaaggtc gagaagggaa agtccaagaa gctcaagtcc gtcaaggagc 5881 tcctcggaat caccatcatg gagcgttcct ccttcgagaa gaacccaatc gacttcctcg 5941 aggccaaggg atacaaggag gtcaagaagg acctcatcat caagctccca aagtactccc 6001 tcttcgagct cgagaacgga cgtaagcgta tgctcgcctc cgccggagag ctccaaaagg 6061 gaaacgagct cgccctccca tccaagtacg tcaacttcct ctacctcgcc tcccactacg 6121 agaagctcaa gggatcccca gaggacaacg agcaaaagca actcttcgtc gagcaacaca 6181 agcactacct cgacgagatc atcgagcaaa tctccgagtt ctccaagcgt gtcatcctcg 6241 ccgacgccaa cctcgacaag gtcctctccg cctacaacaa gcaccgtgac aagccaatcc 6301 gtgagcaagc cgagaacatc atccacctct tcaccctcac caacctcgga gccccagccg 6361 ccttcaagta cttcgacacc accatcgacc gtaagcgtta cacctccacc aaggaggtcc 6421 tcgacgccac cctcatccac caatccatca ccggactcta cgagacccgt atcgacctct 6481 cccaactcgg aggagacgga ggatccggac caccaaagaa gaagcgtaag gtctacccat 6541 acgacgtccc agactacgcc taaactagtg ataaatgcaa aatcctttca agcattccct 6601 tcttctctat cactcttctt tctttttgtc aaaaaattct ctcgctaatt tatttgcttt 6661 tttaatgtta ttattttatg actttttata gtcactgaaa agtttgcatc tgagtgaagt 6721 gaatgctatc aaaatgtgat tctgtctgat gtactttcac aatctctctt caattccatt 6781 ttgaagtgct ttaaacccga aaggttgaga aaaatgcgag cgctcaaata tttgtattgt 6841 gttcgttgag tgacccaaca aaaagaggaa actttattgt gccgccaaga aaaaagtctc 6901 acaactttat tatacatagt tgataattca ctggccgtcg ttttacaacg tcgtgactgg 6961 gaaaaccaaa aaaaactagc aataaaggaa taaaaaactg tacaccttaa aggcgcacac 7021 tctgttttgc aaattttatt tttagttgtg aattttctgc tgagacctga aaatagcaac 7081 tttagtacta ctataatttg tcaacctttt caaaaaaagc atgcaatttt tgagaaactc 7141 ttataaaagc tattattaaa aaaacacctt ttttccaaaa ttattccaca aaaaatatgt 7201 tatgaaatgc ctacaccctc tcacacacac tctttatact actctgtcaa actcacgaga 7261 tgtctgccgc ctcttgtgtt gcccctatat aaacacctcc tattgcgaga tgtcttgatc 7321 ttcgccaact ccagcagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt 7381 atcaacttga aaaagtggca ccgagtcggt gctttttgtg aaatttctgg cgtaatagcg 7441 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc 7501 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc 7561 tcagtgcaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg 7621 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg 7681 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa 7741 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga 7801 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 7861 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 7921 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 7981 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 8041 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 8101 agagttttcg ccccgaagaa cgttttccaa tg //