LOCUS       sequence-314079-        7865 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  this plasmid contains the codon optimized gene encoding Cas9 for C. 
            elegans..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7865)
  AUTHORS   Chen C, Fenk LA, de Bono M
  TITLE     Efficient genome editing in Caenorhabditis elegans by 
            CRISPR-targeted homologous recombination.
  JOURNAL   Nucleic Acids Res. 2013 Sep 5.
  PUBMED    24013562
REFERENCE   2  (bases 1 to 7865)
  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: "Nucleic 
            Acids Res. 2013 Sep 5."
COMMENT     Sequence Label: peft-3::cas-9::tbb-2 3'UTR
FEATURES             Location/Qualifiers
     source          1..7865
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..600
                     /gene="eft-3"
                     /label=eft-3 promoter
                     /note="promoter for the C. elegans elongation factor 
                     1-alpha gene (Mitrovich and Anderson, 2000)"
     protein_bind    609..633
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             647..712
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             713..733
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(734..1774,1826..2812,2864..3922,3974..4984)
                     /codon_start=1
                     /product="S. pyogenes Cas9, codon optimized for C. elegans 
                     with synthetic introns"
                     /label=Cas9
                     /translation="DKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFGSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLADSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFEENPINASRVDAKAILSARLSKSRRLE
                     NLIAQLPGEKRNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNSEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGAYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGHSLHEQIANLAGSPAIKKGILQTVKIVDELVKVMGHK
                     PENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLY
                     YLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPSE
                     EVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVA
                     QILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA
                     VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI
                     TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKES
                     ILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI
                     MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELA
                     LPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADAN
                     LDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDA
                     TLIHQSITGLYETRIDLSQLGGD"
     protein_bind    complement(5063..5087)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      5094..5417
                     /gene="tbb-2"
                     /label=tbb-2 terminator
                     /note="transcription terminator for the C. elegans tubulin 
                     beta-2 gene"
     protein_bind    complement(5421..5441)
                     /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(5449..5466)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5449..5465)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5458..5480)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5793..5815
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5853..5871)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5939..6043
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6044..6112,6113..6904)
                     /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(6262..6281)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7075..7663
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7564..7583
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7790..7812
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7809..7825
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7809..7825
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    7845..7865
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
ORIGIN
        1 gcacctttgg tcttttattg tcaacttcca ttggttcttc cattgtttct gttaaattaa
       61 tgaatttttc ataaaataaa gacattatac aatataaaaa tgaagaattt attgaaaata
      121 aactgccaga gagaaaaagt atgcaacact cccgccgaga gtgtttgaaa tggtgtacgg
      181 tacattttcg tgctaggagt tagatgtgca ggcagcaacg agagggggag agattttttt
      241 gggccttgtg aaattaacgt gagttttctg gtcatctgac taatcatgtt ggttttttgt
      301 tggtttattt tgtttttatc tttgttttta tccagattag gaaatttaaa ttttatgaat
      361 ttataatgag gtcaaacatt cagtcccagc gtttttcctg ttctcactgt ttagtcgaat
      421 ttttatttta ggctttcaac aaatgttcta actgtcttat ttgtgacctc actttttata
      481 tttttttaat ttttaaaaat attagaagtt tctaggataa ttttttcgac ttttattctc
      541 tctaccgtcc gcactcttct tacttttaaa ttaaattgtt tttttttcag ttgggaaaca
      601 ctttgctcac aagtttgtac aaaaaagcag gcttttcaga aaaatggact acaaagatca
      661 cgatggcgac tacaaagatc acgatatcga ctacaaagac gatgacgata aaccaaagaa
      721 gaagcgtaag gtagacaaga agtactccat cggactcgac atcggaacca actccgtcgg
      781 atgggccgtc atcaccgacg actacaaggt cccatccaag aagttcaagg tcctcggaaa
      841 caccgaccgt cactccatca agaagaacct catcggagcc ctcctcttcg gatccggaga
      901 gaccgccgag gccacccgtc tcaagcgtac cgcccgtcgt cgttacaccc gtcgtaagaa
      961 ccgtatctgc tacctccaag agatcttctc caacgagatg gccaaggtcg acgactcctt
     1021 cttccaccgt ctcgaggagt ccttcctcgt cgaggaggac aagaagcacg agcgtcaccc
     1081 aatcttcgga aacatcgtcg acgaggtcgc ctaccacgag aagtacccaa ccatctacca
     1141 cctccgtaag aagctcgccg actccaccga caaggccgac ctccgtctca tctacctcgc
     1201 cctcgcccac atgatcaagt tccgtggaca cttcctcatc gagggagacc tcaacccaga
     1261 caactccgac gtcgacaagc tcttcatcca actcgtccaa atctacaacc aactcttcga
     1321 ggagaaccca atcaacgcct cccgtgtcga cgccaaggcc atcctctccg cccgtctctc
     1381 caagtcccgt cgtctcgaga acctcatcgc ccaactccca ggagagaagc gtaacggact
     1441 cttcggaaac ctcatcgccc tctccctcgg actcacccca aacttcaagt ccaacttcga
     1501 cctcgccgag gacgccaagc tccaactctc caaggacacc tacgacgacg acctcgacaa
     1561 cctcctcgcc caaatcggag accaatacgc cgacctcttc ctcgccgcca agaacctctc
     1621 cgacgccatc ctcctctccg acatcctccg tgtcaactcc gagatcacca aggccccact
     1681 ctccgcctcc atgatcaagc gttacgacga gcaccaccaa gacctcaccc tcctcaaggc
     1741 cctcgtccgt caacaactcc cagagaagta caaggtaagt ttaaacatat atatactaac
     1801 taaccctgat tatttaaatt ttcaggagat cttcttcgac caatccaaga acggatacgc
     1861 cggatacatc gacggaggag cctcccaaga ggagttctac aagttcatca agccaatcct
     1921 cgagaagatg gacggaaccg aggagctcct cgtcaagctc aaccgtgagg acctcctccg
     1981 taagcaacgt accttcgaca acggatccat cccacaccaa atccacctcg gagagctcca
     2041 cgccatcctc cgtcgtcaag aggacttcta cccattcctc aaggacaacc gtgagaagat
     2101 cgagaagatc ctcaccttcc gtatcccata ctacgtcgga ccactcgccc gtggaaactc
     2161 ccgtttcgcc tggatgaccc gtaagtccga ggagaccatc accccatgga acttcgagga
     2221 ggtcgtcgac aagggagcct ccgcccaatc cttcatcgag cgtatgacca acttcgacaa
     2281 gaacctccca aacgagaagg tcctcccaaa gcactccctc ctctacgagt acttcaccgt
     2341 ctacaacgag ctcaccaagg tcaagtacgt caccgaggga atgcgtaagc cagccttcct
     2401 ctccggagag caaaagaagg ccatcgtcga cctcctcttc aagaccaacc gtaaggtcac
     2461 cgtcaagcaa ctcaaggagg actacttcaa gaagatcgag tgcttcgact ccgtcgagat
     2521 ctccggagtc gaggaccgtt tcaacgcctc cctcggagcc taccacgacc tcctcaagat
     2581 catcaaggac aaggacttcc tcgacaacga ggagaacgag gacatcctcg aggacatcgt
     2641 cctcaccctc accctcttcg aggaccgtgg aatgatcgag gagcgtctca agacctacgc
     2701 ccacctcttc gacgacaagg tcatgaagca actcaagcgt cgtcgttaca ccggatgggg
     2761 acgtctctcc cgtaagctca ttaacggaat ccgtgacaag caatccggaa aggtaagttt
     2821 aaacagttcg gtactaacta accatacata tttaaatttt cagaccatcc tcgacttcct
     2881 caagtccgac ggattcgcca accgtaactt catgcaactc atccacgacg actccctcac
     2941 cttcaaggag gacatccaaa aggcccaagt ctccggacaa ggacactccc tccacgagca
     3001 aatcgccaac ctcgccggat ccccagccat caagaaggga atcctccaaa ccgtcaagat
     3061 cgtcgacgag ctcgtcaagg tcatgggaca caagccagag aacatcgtca tcgagatggc
     3121 ccgtgagaac caaaccaccc aaaagggaca aaagaactcc cgtgagcgta tgaagcgtat
     3181 cgaggaggga atcaaggagc tcggatccca aatcctcaag gagcacccag tcgagaacac
     3241 ccaactccaa aacgagaagc tctacctcta ctacctccaa aacggacgtg acatgtacgt
     3301 cgaccaagag ctcgacatca accgtctctc cgactacgac gtcgaccaca tcgtcccaca
     3361 atccttcatc aaggacgact ccatcgacaa caaggtcctc acccgttccg acaagaaccg
     3421 tggaaagtcc gacaacgtcc catccgagga ggtcgtcaag aagatgaaga actactggcg
     3481 tcaactcctc aacgccaagc tcatcaccca acgtaagttc gacaacctca ccaaggccga
     3541 gcgtggagga ctctccgagc tcgacaaggc cggattcatc aagcgtcaac tcgtcgagac
     3601 ccgtcaaatc accaagcacg tcgcccaaat cctcgactcc cgtatgaaca ccaagtacga
     3661 cgagaacgac aagctcatcc gtgaggtcaa ggtcatcacc ctcaagtcca agctcgtctc
     3721 cgacttccgt aaggacttcc aattctacaa ggtccgtgag atcaacaact accaccacgc
     3781 ccacgacgcc tacctcaacg ccgtcgtcgg aaccgccctc atcaagaagt acccaaagct
     3841 cgagtccgag ttcgtctacg gagactacaa ggtctacgac gtccgtaaga tgatcgccaa
     3901 gtccgagcaa gagatcggaa aggtaagttt aaacatgatt ttactaacta actaatctga
     3961 tttaaatttt caggccaccg ccaagtactt cttctactcc aacatcatga acttcttcaa
     4021 gaccgagatc accctcgcca acggagagat ccgtaagcgt ccactcatcg agaccaacgg
     4081 agagaccgga gagatcgtct gggacaaggg acgtgacttc gccaccgtcc gtaaggtcct
     4141 ctccatgcca caagtcaaca tcgtcaagaa gaccgaggtc caaaccggag gattctccaa
     4201 ggagtccatc ctcccaaagc gtaactccga caagctcatc gcccgtaaga aggactggga
     4261 cccaaagaag tacggaggat tcgactcccc aaccgtcgcc tactccgtcc tcgtcgtcgc
     4321 caaggtcgag aagggaaagt ccaagaagct caagtccgtc aaggagctcc tcggaatcac
     4381 catcatggag cgttcctcct tcgagaagaa cccaatcgac ttcctcgagg ccaagggata
     4441 caaggaggtc aagaaggacc tcatcatcaa gctcccaaag tactccctct tcgagctcga
     4501 gaacggacgt aagcgtatgc tcgcctccgc cggagagctc caaaagggaa acgagctcgc
     4561 cctcccatcc aagtacgtca acttcctcta cctcgcctcc cactacgaga agctcaaggg
     4621 atccccagag gacaacgagc aaaagcaact cttcgtcgag caacacaagc actacctcga
     4681 cgagatcatc gagcaaatct ccgagttctc caagcgtgtc atcctcgccg acgccaacct
     4741 cgacaaggtc ctctccgcct acaacaagca ccgtgacaag ccaatccgtg agcaagccga
     4801 gaacatcatc cacctcttca ccctcaccaa cctcggagcc ccagccgcct tcaagtactt
     4861 cgacaccacc atcgaccgta agcgttacac ctccaccaag gaggtcctcg acgccaccct
     4921 catccaccaa tccatcaccg gactctacga gacccgtatc gacctctccc aactcggagg
     4981 agacatgagc cgtagacgaa aagcgaatcc gacaaaactg agtgaaaacg cgaagaagct
     5041 tgccaaggaa gttgaaaatt aaacccagct ttcttgtaca aagtgggcga taaatgcaag
     5101 atcctttcaa gcattccctt cttctctatc actcttcttt ctttttgtca aaaaattctc
     5161 tcgctaattt atttgctttt ttaatgttat tattttatga ctttttatag tcactgaaaa
     5221 gtttgcatct gagtgaagtg aatgctatca aaatgtgatt ctgtctgatg tactttcaca
     5281 atctctcttc aattccattt tgaagtgctt taaacccgaa aggttgagaa aaatgcgagc
     5341 gctcaaatat ttgtattgtg ttcgttgagt gacccaacaa aaagaggaaa ctttattgtg
     5401 ccgccaagaa aaaagtctca caactttatt atacatagtt gataattcac tggccgtcgt
     5461 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     5521 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     5581 gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg
     5641 cggtatttca caccgcatat ggtgcactct cagtgcaatc tgctctgatg ccgcatagtt
     5701 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     5761 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     5821 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     5881 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     5941 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     6001 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     6061 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     6121 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     6181 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     6241 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     6301 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     6361 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     6421 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     6481 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     6541 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     6601 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     6661 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     6721 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     6781 actggggtca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     6841 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6901 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     6961 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     7021 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     7081 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     7141 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     7201 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     7261 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     7321 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     7381 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     7441 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     7501 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     7561 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     7621 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     7681 ctttttacgg ttcctggtct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     7741 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgatacca gcggataaca
     7801 atttcacaca ggaaacagct atgaccatga ttacgccaag ctatcaactt tgtatagaaa
     7861 agttg
//
