LOCUS       sequence-99400-e        8148 bp    DNA     circular SYN 14-APR-2025
DEFINITION  CRISPR/Cas9 plasmid containing sgRNA(F+E); for use in C. elegans.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8148)
  AUTHORS   Ward JD
  TITLE     Rapid and Precise Engineering of the Caenorhabditis elegans Genome 
            with Lethal Mutation Co-conversion and Inactivation of NHEJ Repair.
  JOURNAL   Genetics. 2014 Dec 9. pii: genetics.114.172361.
  PUBMED    25491644
REFERENCE   2  (bases 1 to 8148)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Genetics. 
            2014 Dec 9. pii: genetics.114.172361."
COMMENT     Sequence Label: pJW1219
FEATURES             Location/Qualifiers
     source          1..8148
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(24..43)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      837..1425
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1326..1345
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1552..1574
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1571..1587
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1571..1587
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    1607..1627
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        1628..2227
                     /gene="eft-3"
                     /label=eft-3 promoter
                     /note="promoter for the C. elegans elongation factor 
                     1-alpha gene (Mitrovich and Anderson, 2000)"
     CDS             join(2244..3287,3339..4409,4461..5486,5538..6500)
                     /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             6516..6536
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6537..6563
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      6578..6901
                     /gene="tbb-2"
                     /label=tbb-2 terminator
                     /note="transcription terminator for the C. elegans tubulin 
                     beta-2 gene"
     protein_bind    complement(6905..6925)
                     /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(6933..6950)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6933..6949)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6942..6964)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        6970..7319
                     /label=CeU6 promoter
                     /note="promoter for the C. elegans R07E5.16 U6 snRNA gene 
                     (Dickinson et al., 2013)"
     primer_bind     7700..7722
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7760..7778)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7846..7950
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7951..8019,8020..8148,1..666)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMLMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEY
                     SPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDR
                     WEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRS
                     ALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGA
                     SLIKHW"
ORIGIN
        1 ctcatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
       61 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
      121 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
      181 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
      241 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
      301 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
      361 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
      421 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
      481 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
      541 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
      601 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
      661 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
      721 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
      781 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
      841 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
      901 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
      961 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     1021 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     1081 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     1141 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     1201 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     1261 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     1321 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     1381 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     1441 gcctttttac ggttcctggt cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     1501 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cagcggataa
     1561 caatttcaca caggaaacag ctatgaccat gattacgcca agctatcaac tttgtataga
     1621 aaagttggca cctttggtct tttattgtca acttccattg gttcttccat tgtttctgtt
     1681 aaattaatga atttttcata aaataaagac attatacaat ataaaaatga agaatttatt
     1741 gaaaataaac tgccagagag aaaaagtatg caacactccc gccgagagtg tttgaaatgg
     1801 tgtacggtac attttcgtgc taggagttag atgtgcaggc agcaacgaga gggggagaga
     1861 tttttttggg ccttgtgaaa ttaacgtgag ttttctggtc atctgactaa tcatgttggt
     1921 tttttgttgg tttattttgt ttttatcttt gtttttatcc agattaggaa atttaaattt
     1981 tatgaattta taatgaggtc aaacattcag tcccagcgtt tttcctgttc tcactgttta
     2041 gtcgaatttt tattttaggc tttcaacaaa tgttctaact gtcttatttg tgacctcact
     2101 ttttatattt ttttaatttt taaaaatatt agaagtttct aggataattt tttcgacttt
     2161 tattctctct accgtccgca ctcttcttac ttttaaatta aattgttttt ttttcagttg
     2221 ggaaacactt tgctctagaa aaaatggaca aaaaatacag catcggcctt gacatcggaa
     2281 cgaactccgt cggatgggcc gtcatcaccg acgagtacaa ggtcccatcc aagaagttca
     2341 aggtcctcgg aaacaccgac cgtcactcca tcaagaagaa cctcatcgga gccctcctct
     2401 tcgactccgg agagaccgcc gaggccaccc gtctcaagcg taccgcccgt cgtcgttaca
     2461 cccgtcgtaa gaaccgtatc tgctacctcc aagagatctt ctccaacgag atggccaagg
     2521 tcgacgactc cttcttccac cgtctcgagg agtccttcct cgtcgaggag gacaagaagc
     2581 acgagcgtca cccaatcttc ggaaacatcg tcgacgaggt cgcctaccac gagaagtacc
     2641 caaccatcta ccacctccgt aagaagctcg tcgactccac cgacaaggcc gacctccgtc
     2701 tcatctacct cgccctcgcc cacatgatca agttccgtgg acacttcctc atcgagggag
     2761 acctcaaccc agacaactcc gacgtcgaca agctcttcat ccaactcgtc caaacctaca
     2821 accaactctt cgaggagaac ccaatcaacg cctccggagt cgacgccaag gccatcctct
     2881 ccgcccgtct ctccaagtcc cgtcgtctcg agaacctcat cgcccaactc ccaggagaga
     2941 agaagaacgg actcttcgga aacctcatcg ccctctccct cggactcacc ccaaacttca
     3001 agtccaactt cgacctcgcc gaggacgcca agctccaact ctccaaggac acctacgacg
     3061 acgacctcga caacctcctc gcccaaatcg gagaccaata cgccgacctc ttcctcgccg
     3121 ccaagaacct ctccgacgcc atcctcctct ccgacatcct ccgtgtcaac accgagatca
     3181 ccaaggcccc actctccgcc tccatgatca agcgttacga cgagcaccac caagacctca
     3241 ccctcctcaa ggccctcgtc cgtcaacaac tcccagagaa gtacaaggta agtttaaaca
     3301 tatatatact aactaaccct gattatttaa attttcagga gatcttcttc gaccaatcca
     3361 agaacggata cgccggatac atcgacggag gagcctccca agaggagttc tacaagttca
     3421 tcaagccaat cctcgagaag atggacggaa ccgaggagct cctcgtcaag ctcaaccgtg
     3481 aggacctcct ccgtaagcaa cgtaccttcg acaacggatc catcccacac caaatccacc
     3541 tcggagagct ccacgccatc ctccgtcgtc aagaggactt ctacccattc ctcaaggaca
     3601 accgtgagaa gatcgagaag atcctcacct tccgtatccc atactacgtc ggaccactcg
     3661 cccgtggaaa ctcccgtttc gcctggatga cccgtaagtc cgaggagacc atcaccccat
     3721 ggaacttcga ggaggtcgtc gacaagggag cctccgccca atccttcatc gagcgtatga
     3781 ccaacttcga caagaacctc ccaaacgaga aggtcctccc aaagcactcc ctcctctacg
     3841 agtacttcac cgtctacaac gagctcacca aggtcaagta cgtcaccgag ggaatgcgta
     3901 agccagcctt cctctccgga gagcaaaaga aggccatcgt cgacctcctc ttcaagacca
     3961 accgtaaggt caccgtcaag caactcaagg aggactactt caagaagatc gagtgcttcg
     4021 actccgtcga gatctccgga gtcgaggacc gtttcaacgc ctccctcgga acctaccacg
     4081 acctcctcaa gatcatcaag gacaaggact tcctcgacaa cgaggagaac gaggacatcc
     4141 tcgaggacat cgtcctcacc ctcaccctct tcgaggaccg tgagatgatc gaggagcgtc
     4201 tcaagaccta cgcccacctc ttcgacgaca aggtcatgaa gcagcttaag cgtcgtcgtt
     4261 acaccggatg gggacgtctc tcccgtaagc tcatcaacgg aatccgtgac aagcaatccg
     4321 gaaagaccat cctcgacttc ctcaagtccg acggattcgc caaccgtaac ttcatgcaac
     4381 tcatccacga cgactccctc accttcaagg taagtttaaa cagttcggta ctaactaacc
     4441 atacatattt aaattttcag gaggacatcc aaaaggccca agtctccgga caaggagact
     4501 ccctccacga gcacatcgcc aacctcgccg gatccccagc catcaagaag ggaatcctcc
     4561 aaaccgtcaa ggtcgtcgac gagctcgtca aggtcatggg acgtcacaag ccagagaaca
     4621 tcgtcatcga gatggcccgt gagaaccaaa ccacccaaaa gggacaaaag aactcccgtg
     4681 agcgtatgaa gcgtatcgag gagggaatca aggagctcgg atcccaaatc ctcaaggagc
     4741 acccagtcga gaacacccaa ctccaaaacg agaagctcta cctctactac ctccaaaacg
     4801 gacgtgacat gtacgtcgac caagagctcg acatcaaccg tctctccgac tacgacgtcg
     4861 accacatcgt cccacaatcc ttcctcaagg acgactccat cgacaacaag gtcctcaccc
     4921 gttccgacaa gaaccgtgga aagtccgaca acgtcccatc cgaggaggtc gtcaagaaga
     4981 tgaagaacta ctggcgtcaa ctcctcaacg ccaagctcat cacccaacgt aagttcgaca
     5041 acctcaccaa ggctgagcgt ggaggactct ccgagctcga caaggccgga ttcatcaagc
     5101 gtcaactcgt cgagacccgt caaatcacca agcacgtcgc ccaaatcctc gactcccgta
     5161 tgaacaccaa gtacgacgag aacgacaagc tcatccgtga ggtcaaggtc atcaccctca
     5221 agtccaagct cgtctccgac ttccgtaagg acttccaatt ctacaaggtc cgtgagatca
     5281 acaactacca ccacgcccac gacgcctacc tcaacgccgt agtaggaacc gccctcatca
     5341 agaagtaccc aaagctcgag tccgagttcg tctacggaga ctacaaggtc tacgacgtcc
     5401 gtaagatgat cgccaagtcc gagcaagaga tcggaaaggc caccgccaag tacttcttct
     5461 actccaacat catgaacttc ttcaaggtaa gtttaaacat gattttacta actaactaat
     5521 ctgatttaaa ttttcagacc gagatcaccc tcgccaacgg agagatccgt aagcgtccac
     5581 tcatcgagac caacggagag accggagaga tcgtctggga caagggacgt gacttcgcca
     5641 ccgtccgtaa ggtcctctcc atgccacaag tcaacatcgt caagaagacc gaggtccaaa
     5701 ccggaggatt ctccaaggag tccatcctcc caaagcgtaa ctccgacaag ctcatcgccc
     5761 gtaagaagga ctgggaccca aagaagtacg gaggattcga ctccccaacc gtcgcctact
     5821 ccgtcctcgt cgtcgccaag gtcgagaagg gaaagtccaa gaagctcaag tccgtcaagg
     5881 agctcctcgg aatcaccatc atggagcgtt cctccttcga gaagaaccca atcgacttcc
     5941 tcgaggccaa gggatacaag gaggtcaaga aggacctcat catcaagctc ccaaagtact
     6001 ccctcttcga gctcgagaac ggacgtaagc gtatgctcgc ctccgccgga gagctccaaa
     6061 agggaaacga gctcgccctc ccatccaagt acgtcaactt cctctacctc gcctcccact
     6121 acgagaagct caagggatcc ccagaggaca acgagcaaaa gcaactcttc gtcgagcaac
     6181 acaagcacta cctcgacgag atcatcgagc aaatctccga gttctccaag cgtgtcatcc
     6241 tcgccgacgc caacctcgac aaggtcctct ccgcctacaa caagcaccgt gacaagccaa
     6301 tccgtgagca agccgagaac atcatccacc tcttcaccct caccaacctc ggagccccag
     6361 ccgccttcaa gtacttcgac accaccatcg accgtaagcg ttacacctcc accaaggagg
     6421 tcctcgacgc caccctcatc caccaatcca tcaccggact ctacgagacc cgtatcgacc
     6481 tctcccaact cggaggagac ggaggatccg gaccaccaaa gaagaagcgt aaggtctacc
     6541 catacgacgt cccagactac gcctaaacta gtgataaatg caaaatcctt tcaagcattc
     6601 ccttcttctc tatcactctt ctttcttttt gtcaaaaaat tctctcgcta atttatttgc
     6661 ttttttaatg ttattatttt atgacttttt atagtcactg aaaagtttgc atctgagtga
     6721 agtgaatgct atcaaaatgt gattctgtct gatgtacttt cacaatctct cttcaattcc
     6781 attttgaagt gctttaaacc cgaaaggttg agaaaaatgc gagcgctcaa atatttgtat
     6841 tgtgttcgtt gagtgaccca acaaaaagag gaaactttat tgtgccgcca agaaaaaagt
     6901 ctcacaactt tattatacat agttgataat tcactggccg tcgttttaca acgtcgtgac
     6961 tgggaaaacc aaaaaaaact agcaataaag gaataaaaaa ctgtacacct taaaggcgca
     7021 cactctgttt tgcaaatttt atttttagtt gtgaattttc tgctgagacc tgaaaatagc
     7081 aactttagta ctactataat ttgtcaacct tttcaaaaaa agcatgcaat ttttgagaaa
     7141 ctcttataaa agctattatt aaaaaaacac cttttttcca aaattattcc acaaaaaata
     7201 tgttatgaaa tgcctacacc ctctcacaca cactctttat actactctgt caaactcacg
     7261 agatgtctgc cgcctcttgt gttgccccta tataaacacc tcctattgcg agatgtcttg
     7321 gatggatgtg tagtcaattg tttaagagct atgctggaaa cagcatagca agtttaaata
     7381 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tttgtgaaat
     7441 ttctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     7501 aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     7561 cgcatatggt gcactctcag tgcaatctgc tctgatgccg catagttaag ccagccccga
     7621 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     7681 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     7741 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     7801 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     7861 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     7921 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     7981 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     8041 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     8101 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatg
//
