LOCUS       sequence-178583-        7722 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9 expression in C. elegans.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7722)
  AUTHORS   Dickinson D, Slabodnick M, Chen A, Goldstein B
  TITLE     SapTrap assembly of repair templates for Cas9-triggered homologous 
            recombination with a self-excising cassette.
  JOURNAL   MicroPubl Biol. 2018 May 1;2018:10.17912/W2KT0N. doi: 
            10.17912/W2KT0N.
  PUBMED    32550377
REFERENCE   2  (bases 1 to 7722)
  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: "MicroPubl 
            Biol."; date: "2018-05-1"; volume: "2018:10.17912/W2KT0N. doi"; 
            pages: " 10.17912/W2KT0N"
COMMENT     Sequence Label: pDD121
FEATURES             Location/Qualifiers
     source          1..7722
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     58..80
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     77..93
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     77..93
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    113..133
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        134..733
                     /gene="eft-3"
                     /label=eft-3 promoter
                     /note="promoter for the C. elegans elongation factor 
                     1-alpha gene (Mitrovich and Anderson, 2000)"
     CDS             join(750..1793,1845..2915,2967..3992,4044..5006)
                     /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             5022..5042
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5043..5069
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      5084..5407
                     /gene="tbb-2"
                     /label=tbb-2 terminator
                     /note="transcription terminator for the C. elegans tubulin 
                     beta-2 gene"
     protein_bind    complement(5411..5431)
                     /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(5439..5456)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5439..5455)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5448..5470)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5783..5805
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5843..5861)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5929..6033
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6034..6102,6103..6894)
                     /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(6252..6271)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7065..7653
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7554..7573
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccagc
       61 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct atcaactttg
      121 tatagaaaag ttggcacctt tggtctttta ttgtcaactt ccattggttc ttccattgtt
      181 tctgttaaat taatgaattt ttcataaaat aaagacatta tacaatataa aaatgaagaa
      241 tttattgaaa ataaactgcc agagagaaaa agtatgcaac actcccgccg agagtgtttg
      301 aaatggtgta cggtacattt tcgtgctagg agttagatgt gcaggcagca acgagagggg
      361 gagagatttt tttgggcctt gtgaaattaa cgtgagtttt ctggtcatct gactaatcat
      421 gttggttttt tgttggttta ttttgttttt atctttgttt ttatccagat taggaaattt
      481 aaattttatg aatttataat gaggtcaaac attcagtccc agcgtttttc ctgttctcac
      541 tgtttagtcg aatttttatt ttaggctttc aacaaatgtt ctaactgtct tatttgtgac
      601 ctcacttttt atattttttt aatttttaaa aatattagaa gtttctagga taattttttc
      661 gacttttatt ctctctaccg tccgcactct tcttactttt aaattaaatt gttttttttt
      721 cagttgggaa acactttgct ctagaaaaaa tggacaaaaa atacagcatc ggccttgaca
      781 tcggaacgaa ctccgtcgga tgggccgtca tcaccgacga gtacaaggtc ccatccaaga
      841 agttcaaggt cctcggaaac accgaccgtc actccatcaa gaagaacctc atcggagccc
      901 tcctcttcga ctccggagag accgccgagg ccacccgtct caagcgtacc gcccgtcgtc
      961 gttacacccg tcgtaagaac cgtatctgct acctccaaga gatcttctcc aacgagatgg
     1021 ccaaggtcga cgactccttc ttccaccgtc tcgaggagtc cttcctcgtc gaggaggaca
     1081 agaagcacga gcgtcaccca atcttcggaa acatcgtcga cgaggtcgcc taccacgaga
     1141 agtacccaac catctaccac ctccgtaaga agctcgtcga ctccaccgac aaggccgacc
     1201 tccgtctcat ctacctcgcc ctcgcccaca tgatcaagtt ccgtggacac ttcctcatcg
     1261 agggagacct caacccagac aactccgacg tcgacaagct cttcatccaa ctcgtccaaa
     1321 cctacaacca actcttcgag gagaacccaa tcaacgcctc cggagtcgac gccaaggcca
     1381 tcctctccgc ccgtctctcc aagtcccgtc gtctcgagaa cctcatcgcc caactcccag
     1441 gagagaagaa gaacggactc ttcggaaacc tcatcgccct ctccctcgga ctcaccccaa
     1501 acttcaagtc caacttcgac ctcgccgagg acgccaagct ccaactctcc aaggacacct
     1561 acgacgacga cctcgacaac ctcctcgccc aaatcggaga ccaatacgcc gacctcttcc
     1621 tcgccgccaa gaacctctcc gacgccatcc tcctctccga catcctccgt gtcaacaccg
     1681 agatcaccaa ggccccactc tccgcctcca tgatcaagcg ttacgacgag caccaccaag
     1741 acctcaccct cctcaaggcc ctcgtccgtc aacaactccc agagaagtac aaggtaagtt
     1801 taaacatata tatactaact aaccctgatt atttaaattt tcaggagatc ttcttcgacc
     1861 aatccaagaa cggatacgcc ggatacatcg acggaggagc ctcccaagag gagttctaca
     1921 agttcatcaa gccaatcctc gagaagatgg acggaaccga ggagctcctc gtcaagctca
     1981 accgtgagga cctcctccgt aagcaacgta ccttcgacaa cggatccatc ccacaccaaa
     2041 tccacctcgg agagctccac gccatcctcc gtcgtcaaga ggacttctac ccattcctca
     2101 aggacaaccg tgagaagatc gagaagatcc tcaccttccg tatcccatac tacgtcggac
     2161 cactcgcccg tggaaactcc cgtttcgcct ggatgacccg taagtccgag gagaccatca
     2221 ccccatggaa cttcgaggag gtcgtcgaca agggagcctc cgcccaatcc ttcatcgagc
     2281 gtatgaccaa cttcgacaag aacctcccaa acgagaaggt cctcccaaag cactccctcc
     2341 tctacgagta cttcaccgtc tacaacgagc tcaccaaggt caagtacgtc accgagggaa
     2401 tgcgtaagcc agccttcctc tccggagagc aaaagaaggc catcgtcgac ctcctcttca
     2461 agaccaaccg taaggtcacc gtcaagcaac tcaaggagga ctacttcaag aagatcgagt
     2521 gcttcgactc cgtcgagatc tccggagtcg aggaccgttt caacgcctcc ctcggaacct
     2581 accacgacct cctcaagatc atcaaggaca aggacttcct cgacaacgag gagaacgagg
     2641 acatcctcga ggacatcgtc ctcaccctca ccctcttcga ggaccgtgag atgatcgagg
     2701 agcgtctcaa gacctacgcc cacctcttcg acgacaaggt catgaagcag cttaagcgtc
     2761 gtcgttacac cggatgggga cgtctctccc gtaagctcat caacggaatc cgtgacaagc
     2821 aatccggaaa gaccatcctc gacttcctca agtccgacgg attcgccaac cgtaacttca
     2881 tgcaactcat ccacgacgac tccctcacct tcaaggtaag tttaaacagt tcggtactaa
     2941 ctaaccatac atatttaaat tttcaggagg acatccaaaa ggcccaagtc tccggacaag
     3001 gagactccct ccacgagcac atcgccaacc tcgccggatc cccagccatc aagaagggaa
     3061 tcctccaaac cgtcaaggtc gtcgacgagc tcgtcaaggt catgggacgt cacaagccag
     3121 agaacatcgt catcgagatg gcccgtgaga accaaaccac ccaaaaggga caaaagaact
     3181 cccgtgagcg tatgaagcgt atcgaggagg gaatcaagga gctcggatcc caaatcctca
     3241 aggagcaccc agtcgagaac acccaactcc aaaacgagaa gctctacctc tactacctcc
     3301 aaaacggacg tgacatgtac gtcgaccaag agctcgacat caaccgtctc tccgactacg
     3361 acgtcgacca catcgtccca caatccttcc tcaaggacga ctccatcgac aacaaggtcc
     3421 tcacccgttc cgacaagaac cgtggaaagt ccgacaacgt cccatccgag gaggtcgtca
     3481 agaagatgaa gaactactgg cgtcaactcc tcaacgccaa gctcatcacc caacgtaagt
     3541 tcgacaacct caccaaggct gagcgtggag gactctccga gctcgacaag gccggattca
     3601 tcaagcgtca actcgtcgag acccgtcaaa tcaccaagca cgtcgcccaa atcctcgact
     3661 cccgtatgaa caccaagtac gacgagaacg acaagctcat ccgtgaggtc aaggtcatca
     3721 ccctcaagtc caagctcgtc tccgacttcc gtaaggactt ccaattctac aaggtccgtg
     3781 agatcaacaa ctaccaccac gcccacgacg cctacctcaa cgccgtagta ggaaccgccc
     3841 tcatcaagaa gtacccaaag ctcgagtccg agttcgtcta cggagactac aaggtctacg
     3901 acgtccgtaa gatgatcgcc aagtccgagc aagagatcgg aaaggccacc gccaagtact
     3961 tcttctactc caacatcatg aacttcttca aggtaagttt aaacatgatt ttactaacta
     4021 actaatctga tttaaatttt cagaccgaga tcaccctcgc caacggagag atccgtaagc
     4081 gtccactcat cgagaccaac ggagagaccg gagagatcgt ctgggacaag ggacgtgact
     4141 tcgccacagt ccgtaaggtc ctctccatgc cacaagtcaa catcgtcaag aagaccgagg
     4201 tccaaaccgg aggattctcc aaggagtcca tcctcccaaa gcgtaactcc gacaagctca
     4261 tcgcccgtaa gaaggactgg gacccaaaga agtacggagg attcgactcc ccaaccgtcg
     4321 cctactccgt cctcgtcgtc gccaaggtcg agaagggaaa gtccaagaag ctcaagtccg
     4381 tcaaggagct cctcggaatc accatcatgg agcgttcctc cttcgagaag aacccaatcg
     4441 acttcctcga ggccaaggga tacaaggagg tcaagaagga cctcatcatc aagctcccaa
     4501 agtactccct cttcgagctc gagaacggac gtaagcgtat gctcgcctcc gccggagagc
     4561 tccaaaaggg aaacgagctc gccctcccat ccaagtacgt caacttcctc tacctcgcct
     4621 cccactacga gaagctcaag ggatccccag aggacaacga gcaaaagcaa ctcttcgtcg
     4681 agcaacacaa gcactacctc gacgagatca tcgagcaaat ctccgagttc tccaagcgtg
     4741 tcatcctcgc cgacgccaac ctcgacaagg tcctctccgc ctacaacaag caccgtgaca
     4801 agccaatccg tgagcaagcc gagaacatca tccacctctt caccctcacc aacctcggag
     4861 ccccagccgc cttcaagtac ttcgacacca ccatcgaccg taagcgttac acctccacca
     4921 aggaggtcct cgacgccacc ctcatccacc aatccatcac cggactctac gagacccgta
     4981 tcgacctctc ccaactcgga ggagacggag gatccggacc accaaagaag aagcgtaagg
     5041 tctacccata cgacgtccca gactacgcct aaactagtga taaatgcaaa atcctttcaa
     5101 gcattccctt cttctctatc actcttcttt ctttttgtca aaaaattctc tcgctaattt
     5161 atttgctttt ttaatgttat tattttatga ctttttatag tcactgaaaa gtttgcatct
     5221 gagtgaagtg aatgctatca aaatgtgatt ctgtctgatg tactttcaca atctctcttc
     5281 aattccattt tgaagtgctt taaacccgaa aggttgagaa aaatgcgagc gctcaaatat
     5341 ttgtattgtg ttcgttgagt gacccaacaa aaagaggaaa ctttattgtg ccgccaagaa
     5401 aaaagtctca caactttatt atacatagtt gataattcac tggccgtcgt tttacaacgt
     5461 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     5521 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     5581 ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     5641 caccgcatat ggtgcactct cagtgcaatc tgctctgatg ccgcatagtt aagccagccc
     5701 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     5761 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     5821 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     5881 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     5941 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     6001 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     6061 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     6121 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     6181 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     6241 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     6301 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     6361 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     6421 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     6481 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     6541 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     6601 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     6661 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     6721 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggtca
     6781 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     6841 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     6901 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     6961 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     7021 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     7081 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     7141 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     7201 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     7261 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     7321 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     7381 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     7441 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     7501 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     7561 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     7621 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     7681 ttcctggtct tttgctggcc ttttgctcac atgttctttc ct
//
