LOCUS       sequence-307909-        8132 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9 + sgRNA plasmid that is targeted to the synthetic guide 
            sequence GGACAGTCCTGCCGAGGTGG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8132)
  AUTHORS   Stevenson ZC, Moerdyk-Schauwecker MJ, Jamison B, Phillips PC
  TITLE     Rapid Self-Selecting and Clone-Free Integration of Transgenes into 
            Engineered CRISPR Safe Harbor Locations in Caenorhabditis elegans.
  JOURNAL   G3 (Bethesda). 2020 Aug 19. pii: g3.120.401400. doi: 
            10.1534/g3.120.401400.
  PUBMED    32816924
REFERENCE   2  (bases 1 to 8132)
  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: "G3 
            (Bethesda). 2020 Aug 19. pii: g3.120.401400. doi: 
            10.1534/g3.120.401400."
COMMENT     Sequence Label: pMS79 (eft-3p::Cas9 + sgRNA)
FEATURES             Location/Qualifiers
     source          1..8132
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1..17
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    37..57
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        58..657
                     /gene="eft-3"
                     /label=eft-3 promoter
                     /note="promoter for the C. elegans elongation factor 
                     1-alpha gene (Mitrovich and Anderson, 2000)"
     CDS             join(674..1717,1769..2839,2891..3916,3968..4930)
                     /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             4946..4966
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4967..4993
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      5008..5331
                     /gene="tbb-2"
                     /label=tbb-2 terminator
                     /note="transcription terminator for the C. elegans tubulin 
                     beta-2 gene"
     protein_bind    complement(5335..5355)
                     /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(5363..5380)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5363..5379)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5372..5394)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        5400..5749
                     /label=CeU6 promoter
                     /note="promoter for the C. elegans R07E5.16 U6 snRNA gene 
                     (Dickinson et al., 2013)"
     misc_RNA        5770..5845
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     6117..6139
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6177..6195)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6263..6367
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6368..6436,6437..7228)
                     /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(6586..6605)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7399..7987
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7888..7907
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(8114..8132,1..4)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 caggaaacag ctatgaccat gattacgcca agctatcaac tttgtataga aaagttggca
       61 cctttggtct tttattgtca acttccattg gttcttccat tgtttctgtt aaattaatga
      121 atttttcata aaataaagac attatacaat ataaaaatga agaatttatt gaaaataaac
      181 tgccagagag aaaaagtatg caacactccc gccgagagtg tttgaaatgg tgtacggtac
      241 attttcgtgc taggagttag atgtgcaggc agcaacgaga gggggagaga tttttttggg
      301 ccttgtgaaa ttaacgtgag ttttctggtc atctgactaa tcatgttggt tttttgttgg
      361 tttattttgt ttttatcttt gtttttatcc agattaggaa atttaaattt tatgaattta
      421 taatgaggtc aaacattcag tcccagcgtt tttcctgttc tcactgttta gtcgaatttt
      481 tattttaggc tttcaacaaa tgttctaact gtcttatttg tgacctcact ttttatattt
      541 ttttaatttt taaaaatatt agaagtttct aggataattt tttcgacttt tattctctct
      601 accgtccgca ctcttcttac ttttaaatta aattgttttt ttttcagttg ggaaacactt
      661 tgctctagaa aaaatggaca aaaaatacag catcggcctt gacatcggaa cgaactccgt
      721 cggatgggcc gtcatcaccg acgagtacaa ggtcccatcc aagaagttca aggtcctcgg
      781 aaacaccgac cgtcactcca tcaagaagaa cctcatcgga gccctcctct tcgactccgg
      841 agagaccgcc gaggccaccc gtctcaagcg taccgcccgt cgtcgttaca cccgtcgtaa
      901 gaaccgtatc tgctacctcc aagagatctt ctccaacgag atggccaagg tcgacgactc
      961 cttcttccac cgtctcgagg agtccttcct cgtcgaggag gacaagaagc acgagcgtca
     1021 cccaatcttc ggaaacatcg tcgacgaggt cgcctaccac gagaagtacc caaccatcta
     1081 ccacctccgt aagaagctcg tcgactccac cgacaaggcc gacctccgtc tcatctacct
     1141 cgccctcgcc cacatgatca agttccgtgg acacttcctc atcgagggag acctcaaccc
     1201 agacaactcc gacgtcgaca agctcttcat ccaactcgtc caaacctaca accaactctt
     1261 cgaggagaac ccaatcaacg cctccggagt cgacgccaag gccatcctct ccgcccgtct
     1321 ctccaagtcc cgtcgtctcg agaacctcat cgcccaactc ccaggagaga agaagaacgg
     1381 actcttcgga aacctcatcg ccctctccct cggactcacc ccaaacttca agtccaactt
     1441 cgacctcgcc gaggacgcca agctccaact ctccaaggac acctacgacg acgacctcga
     1501 caacctcctc gcccaaatcg gagaccaata cgccgacctc ttcctcgccg ccaagaacct
     1561 ctccgacgcc atcctcctct ccgacatcct ccgtgtcaac accgagatca ccaaggcccc
     1621 actctccgcc tccatgatca agcgttacga cgagcaccac caagacctca ccctcctcaa
     1681 ggccctcgtc cgtcaacaac tcccagagaa gtacaaggta agtttaaaca tatatatact
     1741 aactaaccct gattatttaa attttcagga gatcttcttc gaccaatcca agaacggata
     1801 cgccggatac atcgacggag gagcctccca agaggagttc tacaagttca tcaagccaat
     1861 cctcgagaag atggacggaa ccgaggagct cctcgtcaag ctcaaccgtg aggacctcct
     1921 ccgtaagcaa cgtaccttcg acaacggatc catcccacac caaatccacc tcggagagct
     1981 ccacgccatc ctccgtcgtc aagaggactt ctacccattc ctcaaggaca accgtgagaa
     2041 gatcgagaag atcctcacct tccgtatccc atactacgtc ggaccactcg cccgtggaaa
     2101 ctcccgtttc gcctggatga cccgtaagtc cgaggagacc atcaccccat ggaacttcga
     2161 ggaggtcgtc gacaagggag cctccgccca atccttcatc gagcgtatga ccaacttcga
     2221 caagaacctc ccaaacgaga aggtcctccc aaagcactcc ctcctctacg agtacttcac
     2281 cgtctacaac gagctcacca aggtcaagta cgtcaccgag ggaatgcgta agccagcctt
     2341 cctctccgga gagcaaaaga aggccatcgt cgacctcctc ttcaagacca accgtaaggt
     2401 caccgtcaag caactcaagg aggactactt caagaagatc gagtgcttcg actccgtcga
     2461 gatctccgga gtcgaggacc gtttcaacgc ctccctcgga acctaccacg acctcctcaa
     2521 gatcatcaag gacaaggact tcctcgacaa cgaggagaac gaggacatcc tcgaggacat
     2581 cgtcctcacc ctcaccctct tcgaggaccg tgagatgatc gaggagcgtc tcaagaccta
     2641 cgcccacctc ttcgacgaca aggtcatgaa gcagcttaag cgtcgtcgtt acaccggatg
     2701 gggacgtctc tcccgtaagc tcatcaacgg aatccgtgac aagcaatccg gaaagaccat
     2761 cctcgacttc ctcaagtccg acggattcgc caaccgtaac ttcatgcaac tcatccacga
     2821 cgactccctc accttcaagg taagtttaaa cagttcggta ctaactaacc atacatattt
     2881 aaattttcag gaggacatcc aaaaggccca agtctccgga caaggagact ccctccacga
     2941 gcacatcgcc aacctcgccg gatccccagc catcaagaag ggaatcctcc aaaccgtcaa
     3001 ggtcgtcgac gagctcgtca aggtcatggg acgtcacaag ccagagaaca tcgtcatcga
     3061 gatggcccgt gagaaccaaa ccacccaaaa gggacaaaag aactcccgtg agcgtatgaa
     3121 gcgtatcgag gagggaatca aggagctcgg atcccaaatc ctcaaggagc acccagtcga
     3181 gaacacccaa ctccaaaacg agaagctcta cctctactac ctccaaaacg gacgtgacat
     3241 gtacgtcgac caagagctcg acatcaaccg tctctccgac tacgacgtcg accacatcgt
     3301 cccacaatcc ttcctcaagg acgactccat cgacaacaag gtcctcaccc gttccgacaa
     3361 gaaccgtgga aagtccgaca acgtcccatc cgaggaggtc gtcaagaaga tgaagaacta
     3421 ctggcgtcaa ctcctcaacg ccaagctcat cacccaacgt aagttcgaca acctcaccaa
     3481 ggctgagcgt ggaggactct ccgagctcga caaggccgga ttcatcaagc gtcaactcgt
     3541 cgagacccgt caaatcacca agcacgtcgc ccaaatcctc gactcccgta tgaacaccaa
     3601 gtacgacgag aacgacaagc tcatccgtga ggtcaaggtc atcaccctca agtccaagct
     3661 cgtctccgac ttccgtaagg acttccaatt ctacaaggtc cgtgagatca acaactacca
     3721 ccacgcccac gacgcctacc tcaacgccgt agtaggaacc gccctcatca agaagtaccc
     3781 aaagctcgag tccgagttcg tctacggaga ctacaaggtc tacgacgtcc gtaagatgat
     3841 cgccaagtcc gagcaagaga tcggaaaggc caccgccaag tacttcttct actccaacat
     3901 catgaacttc ttcaaggtaa gtttaaacat gattttacta actaactaat ctgatttaaa
     3961 ttttcagacc gagatcaccc tcgccaacgg agagatccgt aagcgtccac tcatcgagac
     4021 caacggagag accggagaga tcgtctggga caagggacgt gacttcgcca cagtccgtaa
     4081 ggtcctctcc atgccacaag tcaacatcgt caagaagacc gaggtccaaa ccggaggatt
     4141 ctccaaggag tccatcctcc caaagcgtaa ctccgacaag ctcatcgccc gtaagaagga
     4201 ctgggaccca aagaagtacg gaggattcga ctccccaacc gtcgcctact ccgtcctcgt
     4261 cgtcgccaag gtcgagaagg gaaagtccaa gaagctcaag tccgtcaagg agctcctcgg
     4321 aatcaccatc atggagcgtt cctccttcga gaagaaccca atcgacttcc tcgaggccaa
     4381 gggatacaag gaggtcaaga aggacctcat catcaagctc ccaaagtact ccctcttcga
     4441 gctcgagaac ggacgtaagc gtatgctcgc ctccgccgga gagctccaaa agggaaacga
     4501 gctcgccctc ccatccaagt acgtcaactt cctctacctc gcctcccact acgagaagct
     4561 caagggatcc ccagaggaca acgagcaaaa gcaactcttc gtcgagcaac acaagcacta
     4621 cctcgacgag atcatcgagc aaatctccga gttctccaag cgtgtcatcc tcgccgacgc
     4681 caacctcgac aaggtcctct ccgcctacaa caagcaccgt gacaagccaa tccgtgagca
     4741 agccgagaac atcatccacc tcttcaccct caccaacctc ggagccccag ccgccttcaa
     4801 gtacttcgac accaccatcg accgtaagcg ttacacctcc accaaggagg tcctcgacgc
     4861 caccctcatc caccaatcca tcaccggact ctacgagacc cgtatcgacc tctcccaact
     4921 cggaggagac ggaggatccg gaccaccaaa gaagaagcgt aaggtctacc catacgacgt
     4981 cccagactac gcctaaacta gtgataaatg caaaatcctt tcaagcattc ccttcttctc
     5041 tatcactctt ctttcttttt gtcaaaaaat tctctcgcta atttatttgc ttttttaatg
     5101 ttattatttt atgacttttt atagtcactg aaaagtttgc atctgagtga agtgaatgct
     5161 atcaaaatgt gattctgtct gatgtacttt cacaatctct cttcaattcc attttgaagt
     5221 gctttaaacc cgaaaggttg agaaaaatgc gagcgctcaa atatttgtat tgtgttcgtt
     5281 gagtgaccca acaaaaagag gaaactttat tgtgccgcca agaaaaaagt ctcacaactt
     5341 tattatacat agttgataat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     5401 aaaaaaaact agcaataaag gaataaaaaa ctgtacacct taaaggcgca cactctgttt
     5461 tgcaaatttt atttttagtt gtgaattttc tgctgagacc tgaaaatagc aactttagta
     5521 ctactataat ttgtcaacct tttcaaaaaa agcatgcaat ttttgagaaa ctcttataaa
     5581 agctattatt aaaaaaacac cttttttcca aaattattcc acaaaaaata tgttatgaaa
     5641 tgcctacacc ctctcacaca cactctttat actactctgt caaactcacg agatgtctgc
     5701 cgcctcttgt gttgccccta tataaacacc tcctattgcg agatgtcttg gacagtcctg
     5761 ccgaggtggg ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact
     5821 tgaaaaagtg gcaccgagtc ggtgcttttt gtgaaatttc tggcgtaata gcgaagaggc
     5881 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg
     5941 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtgc
     6001 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc
     6061 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     6121 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct
     6181 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     6241 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     6301 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     6361 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     6421 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     6481 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     6541 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     6601 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     6661 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     6721 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     6781 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     6841 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     6901 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     6961 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     7021 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     7081 tgggtctcgc ggtatcattg cagcactggg gtcagatggt aagccctccc gtatcgtagt
     7141 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     7201 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     7261 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     7321 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     7381 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     7441 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     7501 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     7561 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     7621 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     7681 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     7741 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     7801 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     7861 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     7921 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     7981 atggaaaaac gccagcaacg cggccttttt acggttcctg gtcttttgct ggccttttgc
     8041 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     8101 gtgagctgat accagcggat aacaatttca ca
//
