LOCUS       sequence-65793-e        7799 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Codon optimized Cas9_SV40 NLS with intron.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7799)
  AUTHORS   Friedland AE, Tzur YB, Esvelt KM, Colaiacovo MP, Church GM, Calarco 
            JA
  TITLE     Heritable genome editing in C. elegans via a CRISPR-Cas9 system.
  JOURNAL   Nat Methods. 2013 Jun 30. doi: 10.1038/nmeth.2532.
  PUBMED    23817069
REFERENCE   2  (bases 1 to 7799)
  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 Jun 30. doi: 10.1038/nmeth.2532."
COMMENT     Sequence Label: Peft-3::cas9-SV40_NLS::tbb-2 3'UTR
FEATURES             Location/Qualifiers
     source          1..7799
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        402..1001
                     /gene="eft-3"
                     /label=eft-3 promoter
                     /note="promoter for the C. elegans elongation factor 
                     1-alpha gene (Mitrovich and Anderson, 2000)"
     protein_bind    1010..1034
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(1037..3112,3164..5191)
                     /codon_start=1
                     /product="S. pyogenes Cas9, codon optimized for C. elegans 
                     with a synthetic intron"
                     /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             5204..5224
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      5233..5556
                     /gene="tbb-2"
                     /label=tbb-2 terminator
                     /note="transcription terminator for the C. elegans tubulin 
                     beta-2 gene"
     primer_bind     complement(5578..5594)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5578..5594)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5591..5613)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5602..5618
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5626..5632,5633..5650,5651..5656))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5671..5692
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5809..5826)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5980..6568)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6060..6079)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6739..7530,7531..7599))
                     /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"
     primer_bind     7362..7381
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7600..7704)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7772..7790
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgcacctttg gtcttttatt
      421 gtcaacttcc attggttctt ccattgtttc tgttaaatta atgaattttt cataaaataa
      481 agacattata caatataaaa atgaagaatt tattgaaaat aaactgccag agagaaaaag
      541 tatgcaacac tcccgccgag agtgtttgaa atggtgtacg gtacattttc gtgctaggag
      601 ttagatgtgc aggcagcaac gagaggggga gagatttttt tgggccttgt gaaattaacg
      661 tgagttttct ggtcatctga ctaatcatgt tggttttttg ttggtttatt ttgtttttat
      721 ctttgttttt atccagatta ggaaatttaa attttatgaa tttataatga ggtcaaacat
      781 tcagtcccag cgtttttcct gttctcactg tttagtcgaa tttttatttt aggctttcaa
      841 caaatgttct aactgtctta tttgtgacct cactttttat atttttttaa tttttaaaaa
      901 tattagaagt ttctaggata attttttcga cttttattct ctctaccgtc cgcactcttc
      961 ttacttttaa attaaattgt ttttttttca gttgggaaac actttgctca caagtttgta
     1021 caaaaaagca ggcttaatgg acaagaagta ctccatcgga ctcgacatcg gaaccaactc
     1081 cgtcggatgg gccgtcatca ccgacgagta caaggtccca tccaagaagt tcaaggtcct
     1141 cggaaacacc gaccgtcact ccatcaagaa gaacctcatc ggagccctcc tcttcgactc
     1201 cggagagacc gccgaggcca cccgtctcaa gcgtaccgcc cgtcgtcgtt acacccgtcg
     1261 taagaaccgt atctgctacc tccaggagat cttctccaac gagatggcca aggtcgacga
     1321 ctccttcttc caccgtctcg aggagtcctt cctcgtcgag gaggacaaga agcacgagcg
     1381 tcacccaatc ttcggaaaca tcgtcgacga ggtcgcctac cacgagaagt acccaaccat
     1441 ctaccacctc cgtaagaagc tcgtcgactc caccgacaag gccgacctcc gtctcatcta
     1501 cctcgccctc gcccacatga tcaagttccg tggacacttc ctcatcgagg gagacctcaa
     1561 cccagacaac tccgacgtcg acaagctctt catccagctc gtccagacct acaaccagct
     1621 cttcgaggag aacccaatca acgcctccgg agtcgacgcc aaggccatcc tctccgcccg
     1681 tctctccaag tcccgtcgtc tcgagaacct catcgcccag ctcccaggag agaagaagaa
     1741 cggactcttc ggaaacctca tcgccctctc cctcggactc accccaaact tcaagtccaa
     1801 cttcgacctc gccgaggacg ccaagctcca gctctccaag gacacctacg acgacgacct
     1861 cgacaacctc ctcgcccaga tcggagacca gtacgccgac ctcttcctcg ccgccaagaa
     1921 cctctccgac gccatcctcc tctccgacat cctccgtgtc aacaccgaga tcaccaaggc
     1981 cccactctcc gcctccatga tcaagcgtta cgacgagcac caccaggacc tcaccctcct
     2041 caaggccctc gtccgtcagc agctcccaga gaagtacaag gagatcttct tcgaccagtc
     2101 caagaacgga tacgccggat acatcgacgg aggagcctcc caggaggagt tctacaagtt
     2161 catcaagcca atcctcgaga agatggacgg aaccgaggag ctcctcgtca agctcaaccg
     2221 tgaggacctc ctccgtaagc agcgtacctt cgacaacgga tccatcccac accagatcca
     2281 cctcggagag ctccacgcca tcctccgtcg tcaggaggac ttctacccat tcctcaagga
     2341 caaccgtgag aagatcgaga agatcctcac cttccgtatc ccatactacg tcggaccact
     2401 cgcccgtgga aactcccgtt tcgcctggat gacccgtaag tccgaggaga ccatcacccc
     2461 atggaacttc gaggaggtcg tcgacaaggg agcctccgcc cagtccttca tcgagcgtat
     2521 gaccaacttc gacaagaacc tcccaaacga gaaggtcctc ccaaagcact ccctcctcta
     2581 cgagtacttc accgtctaca acgagctcac caaggtcaag tacgtcaccg agggaatgcg
     2641 taagccagcc ttcctctccg gagagcagaa gaaggccatc gtcgacctcc tcttcaagac
     2701 caaccgtaag gtcaccgtca agcagctcaa ggaggactac ttcaagaaga tcgagtgctt
     2761 cgactccgtc gagatctccg gagtcgagga ccgtttcaac gcctccctcg gaacctacca
     2821 cgacctcctc aagatcatca aggacaagga cttcctcgac aacgaggaga acgaggacat
     2881 cctcgaggac atcgtcctca ccctcaccct cttcgaggac cgtgagatga tcgaggagcg
     2941 tctcaagacc tacgcccacc tcttcgacga caaggtcatg aagcagctca agcgtcgtcg
     3001 ttacaccgga tggggacgtc tctcccgtaa gctcatcaac ggaatccgtg acaagcagtc
     3061 cggaaagacc atcctcgact tcctcaagtc cgacggattc gccaaccgta acgtaagttt
     3121 aaacatgatt ttactaacta actaatctga tttaaatttt cagttcatgc agctcatcca
     3181 cgacgactcc ctcaccttca aggaggacat ccagaaggcc caggtctccg gacagggaga
     3241 ctccctccac gagcacatcg ccaacctcgc cggatcccca gccatcaaga agggaatcct
     3301 ccagaccgtc aaggtcgtcg acgagctcgt caaggtcatg ggacgtcaca agccagagaa
     3361 catcgtcatc gagatggccc gtgagaacca gaccacccag aagggacaga agaactcccg
     3421 tgagcgtatg aagcgtatcg aggagggaat caaggagctc ggatcccaga tcctcaagga
     3481 gcacccagtc gagaacaccc agctccagaa cgagaagctc tacctctact acctccagaa
     3541 cggacgtgac atgtacgtcg accaggagct cgacatcaac cgtctctccg actacgacgt
     3601 cgaccacatc gtcccacagt ccttcctcaa ggacgactcc atcgacaaca aggtcctcac
     3661 ccgttccgac aagaaccgtg gaaagtccga caacgtccca tccgaggagg tcgtcaagaa
     3721 gatgaagaac tactggcgtc agctcctcaa cgccaagctc atcacccagc gtaagttcga
     3781 caacctcacc aaggccgagc gtggaggact ctccgagctc gacaaggccg gattcatcaa
     3841 gcgtcagctc gtcgagaccc gtcagatcac caagcacgtc gcccagatcc tcgactcccg
     3901 tatgaacacc aagtacgacg agaacgacaa gctcatccgt gaggtcaagg tcatcaccct
     3961 caagtccaag ctcgtctccg acttccgtaa ggacttccag ttctacaagg tccgtgagat
     4021 caacaactac caccacgccc acgacgccta cctcaacgcc gtcgtcggaa ccgccctcat
     4081 caagaagtac ccaaagctcg agtccgagtt cgtctacgga gactacaagg tctacgacgt
     4141 ccgtaagatg atcgccaagt ccgagcagga gatcggaaag gccaccgcca agtacttctt
     4201 ctactccaac atcatgaact tcttcaagac cgagatcacc ctcgccaacg gagagatccg
     4261 taagcgtcca ctcatcgaga ccaacggaga gaccggagag atcgtctggg acaagggacg
     4321 tgacttcgcc accgtccgta aggtcctctc catgccacag gtcaacatcg tcaagaagac
     4381 cgaggtccag accggaggat tctccaagga gtccatcctc ccaaagcgta actccgacaa
     4441 gctcatcgcc cgtaagaagg actgggaccc aaagaagtac ggaggattcg actccccaac
     4501 cgtcgcctac tccgtcctcg tcgtcgccaa ggtcgagaag ggaaagtcca agaagctcaa
     4561 gtccgtcaag gagctcctcg gaatcaccat catggagcgt tcctccttcg agaagaaccc
     4621 aatcgacttc ctcgaggcca agggatacaa ggaggtcaag aaggacctca tcatcaagct
     4681 cccaaagtac tccctcttcg agctcgagaa cggacgtaag cgtatgctcg cctccgccgg
     4741 agagctccag aagggaaacg agctcgccct cccatccaag tacgtcaact tcctctacct
     4801 cgcctcccac tacgagaagc tcaagggatc cccagaggac aacgagcaga agcagctctt
     4861 cgtcgagcag cacaagcact acctcgacga gatcatcgag cagatctccg agttctccaa
     4921 gcgtgtcatc ctcgccgacg ccaacctcga caaggtcctc tccgcctaca acaagcaccg
     4981 tgacaagcca atccgtgagc aggccgagaa catcatccac ctcttcaccc tcaccaacct
     5041 cggagcccca gccgccttca agtacttcga caccaccatc gaccgtaagc gttacacctc
     5101 caccaaggag gtcctcgacg ccaccctcat ccaccagtcc atcaccggac tctacgagac
     5161 ccgtatcgac ctctcccagc tcggaggaga ctcccgtgcc gacccaaaga agaagcgtaa
     5221 ggtctgagat aaatgcaaaa tcctttcaag cattcccttc ttctctatca ctcttctttc
     5281 tttttgtcaa aaaattctct cgctaattta tttgcttttt taatgttatt attttatgac
     5341 tttttatagt cactgaaaag tttgcatctg agtgaagtga atgctatcaa aatgtgattc
     5401 tgtctgatgt actttcacaa tctctcttca attccatttt gaagtgcttt aaacccgaaa
     5461 ggttgagaaa aatgcgagcg ctcaaatatt tgtattgtgt tcgttgagtg acccaacaaa
     5521 aagaggaaac tttattgtgc cgccaagaaa aaagtctcaa agcttggcgt aatcatggtc
     5581 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     5641 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     5701 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     5761 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     5821 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     5881 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     5941 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     6001 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     6061 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     6121 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     6181 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     6241 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     6301 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     6361 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     6421 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     6481 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     6541 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     6601 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     6661 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     6721 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     6781 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     6841 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     6901 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     6961 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     7021 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     7081 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     7141 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     7201 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     7261 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     7321 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     7381 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     7441 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     7501 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     7561 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     7621 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     7681 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga
     7741 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtc
//
