LOCUS       pDD162 (Peft-3::Cas9 + Empty sgRNA)               8113 bp ds-DNA     circular SYN 13-NOV-2018
DEFINITION  Cas9 + sgRNA plasmid that can be modified to cleave any Cas9 target 
            site in the C. elegans genome..
ACCESSION   .
VERSION     .
KEYWORDS    pDD162 (Peft-3::Cas9 + Empty sgRNA)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8113)
  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 8113)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 13, 2018 from SnapGene Server 1.1.58
            //www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8113
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     716..738
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     735..751
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     735..751
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    771..791
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        792..1391
                     /gene="eft-3"
                     /label=eft-3 promoter
                     /note="promoter for the C. elegans elongation factor 
                     1-alpha gene (Mitrovich and Anderson, 2000)"
     CDS             join(1408..2451,2503..3573,3625..4650,4702..5664)
                     /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             5680..5700
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5701..5727
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      5742..6065
                     /gene="tbb-2"
                     /label=tbb-2 terminator
                     /note="transcription terminator for the C. elegans tubulin 
                     beta-2 gene"
     protein_bind    complement(6069..6089)
                     /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(6097..6114)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6097..6113)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6106..6128)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        6134..6483
                     /label=CeU6 promoter
                     /note="promoter for the C. elegans R07E5.16 U6 snRNA gene 
                     (Dickinson et al., 2013)"
     misc_RNA        6485..6560
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     6832..6854
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6892..6910)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6978..7082
                     /gene="bla"
                     /label=AmpR promoter
     CDS             7083..7943
                     /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(7301..7320)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggtcttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccagcgg
      721 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat caactttgta
      781 tagaaaagtt ggcacctttg gtcttttatt gtcaacttcc attggttctt ccattgtttc
      841 tgttaaatta atgaattttt cataaaataa agacattata caatataaaa atgaagaatt
      901 tattgaaaat aaactgccag agagaaaaag tatgcaacac tcccgccgag agtgtttgaa
      961 atggtgtacg gtacattttc gtgctaggag ttagatgtgc aggcagcaac gagaggggga
     1021 gagatttttt tgggccttgt gaaattaacg tgagttttct ggtcatctga ctaatcatgt
     1081 tggttttttg ttggtttatt ttgtttttat ctttgttttt atccagatta ggaaatttaa
     1141 attttatgaa tttataatga ggtcaaacat tcagtcccag cgtttttcct gttctcactg
     1201 tttagtcgaa tttttatttt aggctttcaa caaatgttct aactgtctta tttgtgacct
     1261 cactttttat atttttttaa tttttaaaaa tattagaagt ttctaggata attttttcga
     1321 cttttattct ctctaccgtc cgcactcttc ttacttttaa attaaattgt ttttttttca
     1381 gttgggaaac actttgctct agaaaaaatg gacaaaaaat acagcatcgg ccttgacatc
     1441 ggaacgaact ccgtcggatg ggccgtcatc accgacgagt acaaggtccc atccaagaag
     1501 ttcaaggtcc tcggaaacac cgaccgtcac tccatcaaga agaacctcat cggagccctc
     1561 ctcttcgact ccggagagac cgccgaggcc acccgtctca agcgtaccgc ccgtcgtcgt
     1621 tacacccgtc gtaagaaccg tatctgctac ctccaagaga tcttctccaa cgagatggcc
     1681 aaggtcgacg actccttctt ccaccgtctc gaggagtcct tcctcgtcga ggaggacaag
     1741 aagcacgagc gtcacccaat cttcggaaac atcgtcgacg aggtcgccta ccacgagaag
     1801 tacccaacca tctaccacct ccgtaagaag ctcgtcgact ccaccgacaa ggccgacctc
     1861 cgtctcatct acctcgccct cgcccacatg atcaagttcc gtggacactt cctcatcgag
     1921 ggagacctca acccagacaa ctccgacgtc gacaagctct tcatccaact cgtccaaacc
     1981 tacaaccaac tcttcgagga gaacccaatc aacgcctccg gagtcgacgc caaggccatc
     2041 ctctccgccc gtctctccaa gtcccgtcgt ctcgagaacc tcatcgccca actcccagga
     2101 gagaagaaga acggactctt cggaaacctc atcgccctct ccctcggact caccccaaac
     2161 ttcaagtcca acttcgacct cgccgaggac gccaagctcc aactctccaa ggacacctac
     2221 gacgacgacc tcgacaacct cctcgcccaa atcggagacc aatacgccga cctcttcctc
     2281 gccgccaaga acctctccga cgccatcctc ctctccgaca tcctccgtgt caacaccgag
     2341 atcaccaagg ccccactctc cgcctccatg atcaagcgtt acgacgagca ccaccaagac
     2401 ctcaccctcc tcaaggccct cgtccgtcaa caactcccag agaagtacaa ggtaagttta
     2461 aacatatata tactaactaa ccctgattat ttaaattttc aggagatctt cttcgaccaa
     2521 tccaagaacg gatacgccgg atacatcgac ggaggagcct cccaagagga gttctacaag
     2581 ttcatcaagc caatcctcga gaagatggac ggaaccgagg agctcctcgt caagctcaac
     2641 cgtgaggacc tcctccgtaa gcaacgtacc ttcgacaacg gatccatccc acaccaaatc
     2701 cacctcggag agctccacgc catcctccgt cgtcaagagg acttctaccc attcctcaag
     2761 gacaaccgtg agaagatcga gaagatcctc accttccgta tcccatacta cgtcggacca
     2821 ctcgcccgtg gaaactcccg tttcgcctgg atgacccgta agtccgagga gaccatcacc
     2881 ccatggaact tcgaggaggt cgtcgacaag ggagcctccg cccaatcctt catcgagcgt
     2941 atgaccaact tcgacaagaa cctcccaaac gagaaggtcc tcccaaagca ctccctcctc
     3001 tacgagtact tcaccgtcta caacgagctc accaaggtca agtacgtcac cgagggaatg
     3061 cgtaagccag ccttcctctc cggagagcaa aagaaggcca tcgtcgacct cctcttcaag
     3121 accaaccgta aggtcaccgt caagcaactc aaggaggact acttcaagaa gatcgagtgc
     3181 ttcgactccg tcgagatctc cggagtcgag gaccgtttca acgcctccct cggaacctac
     3241 cacgacctcc tcaagatcat caaggacaag gacttcctcg acaacgagga gaacgaggac
     3301 atcctcgagg acatcgtcct caccctcacc ctcttcgagg accgtgagat gatcgaggag
     3361 cgtctcaaga cctacgccca cctcttcgac gacaaggtca tgaagcagct taagcgtcgt
     3421 cgttacaccg gatggggacg tctctcccgt aagctcatca acggaatccg tgacaagcaa
     3481 tccggaaaga ccatcctcga cttcctcaag tccgacggat tcgccaaccg taacttcatg
     3541 caactcatcc acgacgactc cctcaccttc aaggtaagtt taaacagttc ggtactaact
     3601 aaccatacat atttaaattt tcaggaggac atccaaaagg cccaagtctc cggacaagga
     3661 gactccctcc acgagcacat cgccaacctc gccggatccc cagccatcaa gaagggaatc
     3721 ctccaaaccg tcaaggtcgt cgacgagctc gtcaaggtca tgggacgtca caagccagag
     3781 aacatcgtca tcgagatggc ccgtgagaac caaaccaccc aaaagggaca aaagaactcc
     3841 cgtgagcgta tgaagcgtat cgaggaggga atcaaggagc tcggatccca aatcctcaag
     3901 gagcacccag tcgagaacac ccaactccaa aacgagaagc tctacctcta ctacctccaa
     3961 aacggacgtg acatgtacgt cgaccaagag ctcgacatca accgtctctc cgactacgac
     4021 gtcgaccaca tcgtcccaca atccttcctc aaggacgact ccatcgacaa caaggtcctc
     4081 acccgttccg acaagaaccg tggaaagtcc gacaacgtcc catccgagga ggtcgtcaag
     4141 aagatgaaga actactggcg tcaactcctc aacgccaagc tcatcaccca acgtaagttc
     4201 gacaacctca ccaaggctga gcgtggagga ctctccgagc tcgacaaggc cggattcatc
     4261 aagcgtcaac tcgtcgagac ccgtcaaatc accaagcacg tcgcccaaat cctcgactcc
     4321 cgtatgaaca ccaagtacga cgagaacgac aagctcatcc gtgaggtcaa ggtcatcacc
     4381 ctcaagtcca agctcgtctc cgacttccgt aaggacttcc aattctacaa ggtccgtgag
     4441 atcaacaact accaccacgc ccacgacgcc tacctcaacg ccgtagtagg aaccgccctc
     4501 atcaagaagt acccaaagct cgagtccgag ttcgtctacg gagactacaa ggtctacgac
     4561 gtccgtaaga tgatcgccaa gtccgagcaa gagatcggaa aggccaccgc caagtacttc
     4621 ttctactcca acatcatgaa cttcttcaag gtaagtttaa acatgatttt actaactaac
     4681 taatctgatt taaattttca gaccgagatc accctcgcca acggagagat ccgtaagcgt
     4741 ccactcatcg agaccaacgg agagaccgga gagatcgtct gggacaaggg acgtgacttc
     4801 gccacagtcc gtaaggtcct ctccatgcca caagtcaaca tcgtcaagaa gaccgaggtc
     4861 caaaccggag gattctccaa ggagtccatc ctcccaaagc gtaactccga caagctcatc
     4921 gcccgtaaga aggactggga cccaaagaag tacggaggat tcgactcccc aaccgtcgcc
     4981 tactccgtcc tcgtcgtcgc caaggtcgag aagggaaagt ccaagaagct caagtccgtc
     5041 aaggagctcc tcggaatcac catcatggag cgttcctcct tcgagaagaa cccaatcgac
     5101 ttcctcgagg ccaagggata caaggaggtc aagaaggacc tcatcatcaa gctcccaaag
     5161 tactccctct tcgagctcga gaacggacgt aagcgtatgc tcgcctccgc cggagagctc
     5221 caaaagggaa acgagctcgc cctcccatcc aagtacgtca acttcctcta cctcgcctcc
     5281 cactacgaga agctcaaggg atccccagag gacaacgagc aaaagcaact cttcgtcgag
     5341 caacacaagc actacctcga cgagatcatc gagcaaatct ccgagttctc caagcgtgtc
     5401 atcctcgccg acgccaacct cgacaaggtc ctctccgcct acaacaagca ccgtgacaag
     5461 ccaatccgtg agcaagccga gaacatcatc cacctcttca ccctcaccaa cctcggagcc
     5521 ccagccgcct tcaagtactt cgacaccacc atcgaccgta agcgttacac ctccaccaag
     5581 gaggtcctcg acgccaccct catccaccaa tccatcaccg gactctacga gacccgtatc
     5641 gacctctccc aactcggagg agacggagga tccggaccac caaagaagaa gcgtaaggtc
     5701 tacccatacg acgtcccaga ctacgcctaa actagtgata aatgcaaaat cctttcaagc
     5761 attcccttct tctctatcac tcttctttct ttttgtcaaa aaattctctc gctaatttat
     5821 ttgctttttt aatgttatta ttttatgact ttttatagtc actgaaaagt ttgcatctga
     5881 gtgaagtgaa tgctatcaaa atgtgattct gtctgatgta ctttcacaat ctctcttcaa
     5941 ttccattttg aagtgcttta aacccgaaag gttgagaaaa atgcgagcgc tcaaatattt
     6001 gtattgtgtt cgttgagtga cccaacaaaa agaggaaact ttattgtgcc gccaagaaaa
     6061 aagtctcaca actttattat acatagttga taattcactg gccgtcgttt tacaacgtcg
     6121 tgactgggaa aaccaaaaaa aactagcaat aaaggaataa aaaactgtac accttaaagg
     6181 cgcacactct gttttgcaaa ttttattttt agttgtgaat tttctgctga gacctgaaaa
     6241 tagcaacttt agtactacta taatttgtca accttttcaa aaaaagcatg caatttttga
     6301 gaaactctta taaaagctat tattaaaaaa acaccttttt tccaaaatta ttccacaaaa
     6361 aatatgttat gaaatgccta caccctctca cacacactct ttatactact ctgtcaaact
     6421 cacgagatgt ctgccgcctc ttgtgttgcc cctatataaa cacctcctat tgcgagatgt
     6481 cttggtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
     6541 aagtggcacc gagtcggtgc tttttgtgaa atttctggcg taatagcgaa gaggcccgca
     6601 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt
     6661 ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtgcaatct
     6721 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     6781 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     6841 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     6901 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     6961 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     7021 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     7081 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     7141 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     7201 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     7261 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     7321 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     7381 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     7441 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     7501 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     7561 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     7621 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     7681 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     7741 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     7801 ctcgcggtat cattgcagca ctggggtcag atggtaagcc ctcccgtatc gtagttatct
     7861 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     7921 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     7981 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     8041 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     8101 tcaaaggatc ttc
//