LOCUS       sequence-67865-e        8132 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9 + sgRNA plasmid that is targeted to a genomic site near the 
            ttTi5605 Mos1 insertion allele.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8132)
  AUTHORS   Dickinson DJ, Ward JD, Reiner DJ, Goldstein B
  TITLE     Engineering the Caenorhabditis elegans genome using Cas9-triggered 
            homologous recombination.
  JOURNAL   Nat Methods. 2013 Sep 1. doi: 10.1038/nmeth.2641.
  PUBMED    23995389
REFERENCE   2  (bases 1 to 8132)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2013 Sep 1. doi: 10.1038/nmeth.2641."
COMMENT     Sequence Label: pDD122 (Peft-3::Cas9 + ttTi5605 sgRNA)
FEATURES             Location/Qualifiers
     source          1..8132
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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     complement(7565..7584)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     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             join(7935..8003,8004..8132,1..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 tgtcttgata
     7321 tcagtctgtt tcgtaagttt 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 tcagtacaat 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
//
