LOCUS       sequence-426499-        8132 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9 + guide plasmid targeting ChrI split hygromycinR landing pad.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8132)
  AUTHORS   Moerdyk-Schauwecker MJ, Jahahn EK, Munoz ZI, Robinson KJ, Phillips 
            PC
  TITLE     Expansion of the split hygromycin toolkit for transgene insertion in
            Caenorhabditis elegans.
  JOURNAL   MicroPubl Biol. 2024 Jan 29;2024:10.17912/micropub.biology.001091. 
            doi: 10.17912/micropub.biology.001091. eCollection 2024.
  PUBMED    38351905
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: "MicroPubl 
            Biol."; date: "2024-01-29"; volume: 
            "2024:10.17912/micropub.biology.001091. doi"; pages: " 
            10.17912/micropub.biology.001091. eCollection 2024"
COMMENT     Sequence Label: pMS59
FEATURES             Location/Qualifiers
     source          1..8132
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      171..759
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     660..679
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     886..908
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     905..921
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     905..921
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    941..961
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        962..1561
                     /gene="eft-3"
                     /label=eft-3 promoter
                     /note="promoter for the C. elegans elongation factor 
                     1-alpha gene (Mitrovich and Anderson, 2000)"
     CDS             join(1578..2621,2673..3743,3795..4820,4872..5834)
                     /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             5850..5870
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5871..5897
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      5912..6235
                     /gene="tbb-2"
                     /label=tbb-2 terminator
                     /note="transcription terminator for the C. elegans tubulin 
                     beta-2 gene"
     protein_bind    complement(6239..6259)
                     /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(6267..6284)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6267..6283)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6276..6298)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        6304..6653
                     /label=CeU6 promoter
                     /note="promoter for the C. elegans R07E5.16 U6 snRNA gene 
                     (Dickinson et al., 2013)"
     misc_RNA        6674..6749
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     7021..7043
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7081..7099)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7167..7271
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7272..7340,7341..8132)
                     /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(7490..7509)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
       61 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
      121 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
      181 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
      241 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
      301 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
      361 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
      421 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
      481 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
      541 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
      601 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
      661 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
      721 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
      781 ttacggttcc tggtcttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
      841 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccagcgg ataacaattt
      901 cacacaggaa acagctatga ccatgattac gccaagctat caactttgta tagaaaagtt
      961 ggcacctttg gtcttttatt gtcaacttcc attggttctt ccattgtttc tgttaaatta
     1021 atgaattttt cataaaataa agacattata caatataaaa atgaagaatt tattgaaaat
     1081 aaactgccag agagaaaaag tatgcaacac tcccgccgag agtgtttgaa atggtgtacg
     1141 gtacattttc gtgctaggag ttagatgtgc aggcagcaac gagaggggga gagatttttt
     1201 tgggccttgt gaaattaacg tgagttttct ggtcatctga ctaatcatgt tggttttttg
     1261 ttggtttatt ttgtttttat ctttgttttt atccagatta ggaaatttaa attttatgaa
     1321 tttataatga ggtcaaacat tcagtcccag cgtttttcct gttctcactg tttagtcgaa
     1381 tttttatttt aggctttcaa caaatgttct aactgtctta tttgtgacct cactttttat
     1441 atttttttaa tttttaaaaa tattagaagt ttctaggata attttttcga cttttattct
     1501 ctctaccgtc cgcactcttc ttacttttaa attaaattgt ttttttttca gttgggaaac
     1561 actttgctct agaaaaaatg gacaaaaaat acagcatcgg ccttgacatc ggaacgaact
     1621 ccgtcggatg ggccgtcatc accgacgagt acaaggtccc atccaagaag ttcaaggtcc
     1681 tcggaaacac cgaccgtcac tccatcaaga agaacctcat cggagccctc ctcttcgact
     1741 ccggagagac cgccgaggcc acccgtctca agcgtaccgc ccgtcgtcgt tacacccgtc
     1801 gtaagaaccg tatctgctac ctccaagaga tcttctccaa cgagatggcc aaggtcgacg
     1861 actccttctt ccaccgtctc gaggagtcct tcctcgtcga ggaggacaag aagcacgagc
     1921 gtcacccaat cttcggaaac atcgtcgacg aggtcgccta ccacgagaag tacccaacca
     1981 tctaccacct ccgtaagaag ctcgtcgact ccaccgacaa ggccgacctc cgtctcatct
     2041 acctcgccct cgcccacatg atcaagttcc gtggacactt cctcatcgag ggagacctca
     2101 acccagacaa ctccgacgtc gacaagctct tcatccaact cgtccaaacc tacaaccaac
     2161 tcttcgagga gaacccaatc aacgcctccg gagtcgacgc caaggccatc ctctccgccc
     2221 gtctctccaa gtcccgtcgt ctcgagaacc tcatcgccca actcccagga gagaagaaga
     2281 acggactctt cggaaacctc atcgccctct ccctcggact caccccaaac ttcaagtcca
     2341 acttcgacct cgccgaggac gccaagctcc aactctccaa ggacacctac gacgacgacc
     2401 tcgacaacct cctcgcccaa atcggagacc aatacgccga cctcttcctc gccgccaaga
     2461 acctctccga cgccatcctc ctctccgaca tcctccgtgt caacaccgag atcaccaagg
     2521 ccccactctc cgcctccatg atcaagcgtt acgacgagca ccaccaagac ctcaccctcc
     2581 tcaaggccct cgtccgtcaa caactcccag agaagtacaa ggtaagttta aacatatata
     2641 tactaactaa ccctgattat ttaaattttc aggagatctt cttcgaccaa tccaagaacg
     2701 gatacgccgg atacatcgac ggaggagcct cccaagagga gttctacaag ttcatcaagc
     2761 caatcctcga gaagatggac ggaaccgagg agctcctcgt caagctcaac cgtgaggacc
     2821 tcctccgtaa gcaacgtacc ttcgacaacg gatccatccc acaccaaatc cacctcggag
     2881 agctccacgc catcctccgt cgtcaagagg acttctaccc attcctcaag gacaaccgtg
     2941 agaagatcga gaagatcctc accttccgta tcccatacta cgtcggacca ctcgcccgtg
     3001 gaaactcccg tttcgcctgg atgacccgta agtccgagga gaccatcacc ccatggaact
     3061 tcgaggaggt cgtcgacaag ggagcctccg cccaatcctt catcgagcgt atgaccaact
     3121 tcgacaagaa cctcccaaac gagaaggtcc tcccaaagca ctccctcctc tacgagtact
     3181 tcaccgtcta caacgagctc accaaggtca agtacgtcac cgagggaatg cgtaagccag
     3241 ccttcctctc cggagagcaa aagaaggcca tcgtcgacct cctcttcaag accaaccgta
     3301 aggtcaccgt caagcaactc aaggaggact acttcaagaa gatcgagtgc ttcgactccg
     3361 tcgagatctc cggagtcgag gaccgtttca acgcctccct cggaacctac cacgacctcc
     3421 tcaagatcat caaggacaag gacttcctcg acaacgagga gaacgaggac atcctcgagg
     3481 acatcgtcct caccctcacc ctcttcgagg accgtgagat gatcgaggag cgtctcaaga
     3541 cctacgccca cctcttcgac gacaaggtca tgaagcagct taagcgtcgt cgttacaccg
     3601 gatggggacg tctctcccgt aagctcatca acggaatccg tgacaagcaa tccggaaaga
     3661 ccatcctcga cttcctcaag tccgacggat tcgccaaccg taacttcatg caactcatcc
     3721 acgacgactc cctcaccttc aaggtaagtt taaacagttc ggtactaact aaccatacat
     3781 atttaaattt tcaggaggac atccaaaagg cccaagtctc cggacaagga gactccctcc
     3841 acgagcacat cgccaacctc gccggatccc cagccatcaa gaagggaatc ctccaaaccg
     3901 tcaaggtcgt cgacgagctc gtcaaggtca tgggacgtca caagccagag aacatcgtca
     3961 tcgagatggc ccgtgagaac caaaccaccc aaaagggaca aaagaactcc cgtgagcgta
     4021 tgaagcgtat cgaggaggga atcaaggagc tcggatccca aatcctcaag gagcacccag
     4081 tcgagaacac ccaactccaa aacgagaagc tctacctcta ctacctccaa aacggacgtg
     4141 acatgtacgt cgaccaagag ctcgacatca accgtctctc cgactacgac gtcgaccaca
     4201 tcgtcccaca atccttcctc aaggacgact ccatcgacaa caaggtcctc acccgttccg
     4261 acaagaaccg tggaaagtcc gacaacgtcc catccgagga ggtcgtcaag aagatgaaga
     4321 actactggcg tcaactcctc aacgccaagc tcatcaccca acgtaagttc gacaacctca
     4381 ccaaggctga gcgtggagga ctctccgagc tcgacaaggc cggattcatc aagcgtcaac
     4441 tcgtcgagac ccgtcaaatc accaagcacg tcgcccaaat cctcgactcc cgtatgaaca
     4501 ccaagtacga cgagaacgac aagctcatcc gtgaggtcaa ggtcatcacc ctcaagtcca
     4561 agctcgtctc cgacttccgt aaggacttcc aattctacaa ggtccgtgag atcaacaact
     4621 accaccacgc ccacgacgcc tacctcaacg ccgtagtagg aaccgccctc atcaagaagt
     4681 acccaaagct cgagtccgag ttcgtctacg gagactacaa ggtctacgac gtccgtaaga
     4741 tgatcgccaa gtccgagcaa gagatcggaa aggccaccgc caagtacttc ttctactcca
     4801 acatcatgaa cttcttcaag gtaagtttaa acatgatttt actaactaac taatctgatt
     4861 taaattttca gaccgagatc accctcgcca acggagagat ccgtaagcgt ccactcatcg
     4921 agaccaacgg agagaccgga gagatcgtct gggacaaggg acgtgacttc gccacagtcc
     4981 gtaaggtcct ctccatgcca caagtcaaca tcgtcaagaa gaccgaggtc caaaccggag
     5041 gattctccaa ggagtccatc ctcccaaagc gtaactccga caagctcatc gcccgtaaga
     5101 aggactggga cccaaagaag tacggaggat tcgactcccc aaccgtcgcc tactccgtcc
     5161 tcgtcgtcgc caaggtcgag aagggaaagt ccaagaagct caagtccgtc aaggagctcc
     5221 tcggaatcac catcatggag cgttcctcct tcgagaagaa cccaatcgac ttcctcgagg
     5281 ccaagggata caaggaggtc aagaaggacc tcatcatcaa gctcccaaag tactccctct
     5341 tcgagctcga gaacggacgt aagcgtatgc tcgcctccgc cggagagctc caaaagggaa
     5401 acgagctcgc cctcccatcc aagtacgtca acttcctcta cctcgcctcc cactacgaga
     5461 agctcaaggg atccccagag gacaacgagc aaaagcaact cttcgtcgag caacacaagc
     5521 actacctcga cgagatcatc gagcaaatct ccgagttctc caagcgtgtc atcctcgccg
     5581 acgccaacct cgacaaggtc ctctccgcct acaacaagca ccgtgacaag ccaatccgtg
     5641 agcaagccga gaacatcatc cacctcttca ccctcaccaa cctcggagcc ccagccgcct
     5701 tcaagtactt cgacaccacc atcgaccgta agcgttacac ctccaccaag gaggtcctcg
     5761 acgccaccct catccaccaa tccatcaccg gactctacga gacccgtatc gacctctccc
     5821 aactcggagg agacggagga tccggaccac caaagaagaa gcgtaaggtc tacccatacg
     5881 acgtcccaga ctacgcctaa actagtgata aatgcaaaat cctttcaagc attcccttct
     5941 tctctatcac tcttctttct ttttgtcaaa aaattctctc gctaatttat ttgctttttt
     6001 aatgttatta ttttatgact ttttatagtc actgaaaagt ttgcatctga gtgaagtgaa
     6061 tgctatcaaa atgtgattct gtctgatgta ctttcacaat ctctcttcaa ttccattttg
     6121 aagtgcttta aacccgaaag gttgagaaaa atgcgagcgc tcaaatattt gtattgtgtt
     6181 cgttgagtga cccaacaaaa agaggaaact ttattgtgcc gccaagaaaa aagtctcaca
     6241 actttattat acatagttga taattcactg gccgtcgttt tacaacgtcg tgactgggaa
     6301 aaccaaaaaa aactagcaat aaaggaataa aaaactgtac accttaaagg cgcacactct
     6361 gttttgcaaa ttttattttt agttgtgaat tttctgctga gacctgaaaa tagcaacttt
     6421 agtactacta taatttgtca accttttcaa aaaaagcatg caatttttga gaaactctta
     6481 taaaagctat tattaaaaaa acaccttttt tccaaaatta ttccacaaaa aatatgttat
     6541 gaaatgccta caccctctca cacacactct ttatactact ctgtcaaact cacgagatgt
     6601 ctgccgcctc ttgtgttgcc cctatataaa cacctcctat tgcgagatgt cttgtttgag
     6661 tagagcactc agggttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
     6721 aacttgaaaa agtggcaccg agtcggtgct ttttgtgaaa tttctggcgt aatagcgaag
     6781 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga
     6841 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca
     6901 gtgcaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
     6961 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     7021 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
     7081 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     7141 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     7201 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     7261 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     7321 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     7381 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     7441 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     7501 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     7561 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     7621 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     7681 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     7741 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     7801 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     7861 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     7921 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     7981 agcgtgggtc tcgcggtatc attgcagcac tggggtcaga tggtaagccc tcccgtatcg
     8041 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     8101 agataggtgc ctcactgatt aagcattggt aa
//
