LOCUS       Exported                8145 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  CRISPR/Cas9 plasmid containing sgRNA(F+E); for use in C. elegans.
ACCESSION   .
VERSION     .
KEYWORDS    pJW1219
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8145)
  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 8145)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8145
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      61..649
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     550..569
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     776..798
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     795..811
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     795..811
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    831..851
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        852..1451
                     /gene="eft-3"
                     /label=eft-3 promoter
                     /note="promoter for the C. elegans elongation factor 
                     1-alpha gene (Mitrovich and Anderson, 2000)"
     CDS             join(1468..2511,2563..3633,3685..4710,4762..5724)
                     /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             5740..5760
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5761..5787
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      5802..6125
                     /gene="tbb-2"
                     /label=tbb-2 terminator
                     /note="transcription terminator for the C. elegans tubulin 
                     beta-2 gene"
     protein_bind    complement(6129..6149)
                     /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(6157..6174)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6157..6173)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6166..6188)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        6194..6543
                     /label=CeU6 promoter
                     /note="promoter for the C. elegans R07E5.16 U6 snRNA gene 
                     (Dickinson et al., 2013)"
     primer_bind     6924..6946
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6984..7002)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7070..7174
                     /gene="bla"
                     /label=AmpR promoter
     CDS             7175..8035
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGSDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(7393..7412)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
       61 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
      121 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      181 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      241 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      301 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      361 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      421 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      481 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      541 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      601 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      661 cgcggccttt ttacggttcc tggtcttttg ctggcctttt gctcacatgt tctttcctgc
      721 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccagcgg
      781 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat caactttgta
      841 tagaaaagtt ggcacctttg gtcttttatt gtcaacttcc attggttctt ccattgtttc
      901 tgttaaatta atgaattttt cataaaataa agacattata caatataaaa atgaagaatt
      961 tattgaaaat aaactgccag agagaaaaag tatgcaacac tcccgccgag agtgtttgaa
     1021 atggtgtacg gtacattttc gtgctaggag ttagatgtgc aggcagcaac gagaggggga
     1081 gagatttttt tgggccttgt gaaattaacg tgagttttct ggtcatctga ctaatcatgt
     1141 tggttttttg ttggtttatt ttgtttttat ctttgttttt atccagatta ggaaatttaa
     1201 attttatgaa tttataatga ggtcaaacat tcagtcccag cgtttttcct gttctcactg
     1261 tttagtcgaa tttttatttt aggctttcaa caaatgttct aactgtctta tttgtgacct
     1321 cactttttat atttttttaa tttttaaaaa tattagaagt ttctaggata attttttcga
     1381 cttttattct ctctaccgtc cgcactcttc ttacttttaa attaaattgt ttttttttca
     1441 gttgggaaac actttgctct agaaaaaatg gacaaaaaat acagcatcgg ccttgacatc
     1501 ggaacgaact ccgtcggatg ggccgtcatc accgacgagt acaaggtccc atccaagaag
     1561 ttcaaggtcc tcggaaacac cgaccgtcac tccatcaaga agaacctcat cggagccctc
     1621 ctcttcgact ccggagagac cgccgaggcc acccgtctca agcgtaccgc ccgtcgtcgt
     1681 tacacccgtc gtaagaaccg tatctgctac ctccaagaga tcttctccaa cgagatggcc
     1741 aaggtcgacg actccttctt ccaccgtctc gaggagtcct tcctcgtcga ggaggacaag
     1801 aagcacgagc gtcacccaat cttcggaaac atcgtcgacg aggtcgccta ccacgagaag
     1861 tacccaacca tctaccacct ccgtaagaag ctcgtcgact ccaccgacaa ggccgacctc
     1921 cgtctcatct acctcgccct cgcccacatg atcaagttcc gtggacactt cctcatcgag
     1981 ggagacctca acccagacaa ctccgacgtc gacaagctct tcatccaact cgtccaaacc
     2041 tacaaccaac tcttcgagga gaacccaatc aacgcctccg gagtcgacgc caaggccatc
     2101 ctctccgccc gtctctccaa gtcccgtcgt ctcgagaacc tcatcgccca actcccagga
     2161 gagaagaaga acggactctt cggaaacctc atcgccctct ccctcggact caccccaaac
     2221 ttcaagtcca acttcgacct cgccgaggac gccaagctcc aactctccaa ggacacctac
     2281 gacgacgacc tcgacaacct cctcgcccaa atcggagacc aatacgccga cctcttcctc
     2341 gccgccaaga acctctccga cgccatcctc ctctccgaca tcctccgtgt caacaccgag
     2401 atcaccaagg ccccactctc cgcctccatg atcaagcgtt acgacgagca ccaccaagac
     2461 ctcaccctcc tcaaggccct cgtccgtcaa caactcccag agaagtacaa ggtaagttta
     2521 aacatatata tactaactaa ccctgattat ttaaattttc aggagatctt cttcgaccaa
     2581 tccaagaacg gatacgccgg atacatcgac ggaggagcct cccaagagga gttctacaag
     2641 ttcatcaagc caatcctcga gaagatggac ggaaccgagg agctcctcgt caagctcaac
     2701 cgtgaggacc tcctccgtaa gcaacgtacc ttcgacaacg gatccatccc acaccaaatc
     2761 cacctcggag agctccacgc catcctccgt cgtcaagagg acttctaccc attcctcaag
     2821 gacaaccgtg agaagatcga gaagatcctc accttccgta tcccatacta cgtcggacca
     2881 ctcgcccgtg gaaactcccg tttcgcctgg atgacccgta agtccgagga gaccatcacc
     2941 ccatggaact tcgaggaggt cgtcgacaag ggagcctccg cccaatcctt catcgagcgt
     3001 atgaccaact tcgacaagaa cctcccaaac gagaaggtcc tcccaaagca ctccctcctc
     3061 tacgagtact tcaccgtcta caacgagctc accaaggtca agtacgtcac cgagggaatg
     3121 cgtaagccag ccttcctctc cggagagcaa aagaaggcca tcgtcgacct cctcttcaag
     3181 accaaccgta aggtcaccgt caagcaactc aaggaggact acttcaagaa gatcgagtgc
     3241 ttcgactccg tcgagatctc cggagtcgag gaccgtttca acgcctccct cggaacctac
     3301 cacgacctcc tcaagatcat caaggacaag gacttcctcg acaacgagga gaacgaggac
     3361 atcctcgagg acatcgtcct caccctcacc ctcttcgagg accgtgagat gatcgaggag
     3421 cgtctcaaga cctacgccca cctcttcgac gacaaggtca tgaagcagct taagcgtcgt
     3481 cgttacaccg gatggggacg tctctcccgt aagctcatca acggaatccg tgacaagcaa
     3541 tccggaaaga ccatcctcga cttcctcaag tccgacggat tcgccaaccg taacttcatg
     3601 caactcatcc acgacgactc cctcaccttc aaggtaagtt taaacagttc ggtactaact
     3661 aaccatacat atttaaattt tcaggaggac atccaaaagg cccaagtctc cggacaagga
     3721 gactccctcc acgagcacat cgccaacctc gccggatccc cagccatcaa gaagggaatc
     3781 ctccaaaccg tcaaggtcgt cgacgagctc gtcaaggtca tgggacgtca caagccagag
     3841 aacatcgtca tcgagatggc ccgtgagaac caaaccaccc aaaagggaca aaagaactcc
     3901 cgtgagcgta tgaagcgtat cgaggaggga atcaaggagc tcggatccca aatcctcaag
     3961 gagcacccag tcgagaacac ccaactccaa aacgagaagc tctacctcta ctacctccaa
     4021 aacggacgtg acatgtacgt cgaccaagag ctcgacatca accgtctctc cgactacgac
     4081 gtcgaccaca tcgtcccaca atccttcctc aaggacgact ccatcgacaa caaggtcctc
     4141 acccgttccg acaagaaccg tggaaagtcc gacaacgtcc catccgagga ggtcgtcaag
     4201 aagatgaaga actactggcg tcaactcctc aacgccaagc tcatcaccca acgtaagttc
     4261 gacaacctca ccaaggctga gcgtggagga ctctccgagc tcgacaaggc cggattcatc
     4321 aagcgtcaac tcgtcgagac ccgtcaaatc accaagcacg tcgcccaaat cctcgactcc
     4381 cgtatgaaca ccaagtacga cgagaacgac aagctcatcc gtgaggtcaa ggtcatcacc
     4441 ctcaagtcca agctcgtctc cgacttccgt aaggacttcc aattctacaa ggtccgtgag
     4501 atcaacaact accaccacgc ccacgacgcc tacctcaacg ccgtagtagg aaccgccctc
     4561 atcaagaagt acccaaagct cgagtccgag ttcgtctacg gagactacaa ggtctacgac
     4621 gtccgtaaga tgatcgccaa gtccgagcaa gagatcggaa aggccaccgc caagtacttc
     4681 ttctactcca acatcatgaa cttcttcaag gtaagtttaa acatgatttt actaactaac
     4741 taatctgatt taaattttca gaccgagatc accctcgcca acggagagat ccgtaagcgt
     4801 ccactcatcg agaccaacgg agagaccgga gagatcgtct gggacaaggg acgtgacttc
     4861 gccacagtcc gtaaggtcct ctccatgcca caagtcaaca tcgtcaagaa gaccgaggtc
     4921 caaaccggag gattctccaa ggagtccatc ctcccaaagc gtaactccga caagctcatc
     4981 gcccgtaaga aggactggga cccaaagaag tacggaggat tcgactcccc aaccgtcgcc
     5041 tactccgtcc tcgtcgtcgc caaggtcgag aagggaaagt ccaagaagct caagtccgtc
     5101 aaggagctcc tcggaatcac catcatggag cgttcctcct tcgagaagaa cccaatcgac
     5161 ttcctcgagg ccaagggata caaggaggtc aagaaggacc tcatcatcaa gctcccaaag
     5221 tactccctct tcgagctcga gaacggacgt aagcgtatgc tcgcctccgc cggagagctc
     5281 caaaagggaa acgagctcgc cctcccatcc aagtacgtca acttcctcta cctcgcctcc
     5341 cactacgaga agctcaaggg atccccagag gacaacgagc aaaagcaact cttcgtcgag
     5401 caacacaagc actacctcga cgagatcatc gagcaaatct ccgagttctc caagcgtgtc
     5461 atcctcgccg acgccaacct cgacaaggtc ctctccgcct acaacaagca ccgtgacaag
     5521 ccaatccgtg agcaagccga gaacatcatc cacctcttca ccctcaccaa cctcggagcc
     5581 ccagccgcct tcaagtactt cgacaccacc atcgaccgta agcgttacac ctccaccaag
     5641 gaggtcctcg acgccaccct catccaccaa tccatcaccg gactctacga gacccgtatc
     5701 gacctctccc aactcggagg agacggagga tccggaccac caaagaagaa gcgtaaggtc
     5761 tacccatacg acgtcccaga ctacgcctaa actagtgata aatgcaaaat cctttcaagc
     5821 attcccttct tctctatcac tcttctttct ttttgtcaaa aaattctctc gctaatttat
     5881 ttgctttttt aatgttatta ttttatgact ttttatagtc actgaaaagt ttgcatctga
     5941 gtgaagtgaa tgctatcaaa atgtgattct gtctgatgta ctttcacaat ctctcttcaa
     6001 ttccattttg aagtgcttta aacccgaaag gttgagaaaa atgcgagcgc tcaaatattt
     6061 gtattgtgtt cgttgagtga cccaacaaaa agaggaaact ttattgtgcc gccaagaaaa
     6121 aagtctcaca actttattat acatagttga taattcactg gccgtcgttt tacaacgtcg
     6181 tgactgggaa aaccaaaaaa aactagcaat aaaggaataa aaaactgtac accttaaagg
     6241 cgcacactct gttttgcaaa ttttattttt agttgtgaat tttctgctga gacctgaaaa
     6301 tagcaacttt agtactacta taatttgtca accttttcaa aaaaagcatg caatttttga
     6361 gaaactctta taaaagctat tattaaaaaa acaccttttt tccaaaatta ttccacaaaa
     6421 aatatgttat gaaatgccta caccctctca cacacactct ttatactact ctgtcaaact
     6481 cacgagatgt ctgccgcctc ttgtgttgcc cctatataaa cacctcctat tgcgagatgt
     6541 cttggatgga tgtgtagtca attgtttaag agctatgctg gaaacagcat agcaagttta
     6601 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttttgtg
     6661 aaatttctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     6721 cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     6781 acaccgcata tggtgcactc tcagtgcaat ctgctctgat gccgcatagt taagccagcc
     6841 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     6901 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     6961 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     7021 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     7081 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     7141 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     7201 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     7261 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     7321 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     7381 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     7441 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     7501 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     7561 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     7621 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     7681 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     7741 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     7801 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     7861 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggtc
     7921 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     7981 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     8041 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     8101 gatctaggtg aagatccttt ttgataatct catgaccaaa atccc
//
