LOCUS       sequence-318017-        6224 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Three-fragment Gateway compatible flourophore for expression in C. 
            elegans.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6224)
  AUTHORS   Aljohani MD, El Mouridi S, Priyadarshini M, Vargas-Velazquez AM, 
            Frokjaer-Jensen C
  TITLE     Engineering rules that minimize germline silencing of transgenes in 
            simple extrachromosomal arrays in C. elegans.
  JOURNAL   Nat Commun. 2020 Dec 9;11(1):6300. doi: 10.1038/s41467-020-19898-0.
  PUBMED    33298957
REFERENCE   2  (bases 1 to 6224)
  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 
            Commun."; date: "2020-12-9"; volume: "11(1):6300. doi"; pages: " 
            10.1038/s41467-020-19898-0"
COMMENT     Sequence Label: pCFJ2452 - [1-2] ENTR - Fluor -
            ce-mNeon(PATCs...
FEATURES             Location/Qualifiers
     source          1..6224
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    33..132
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             141..161
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      1364..1373
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3734..3805
                     /codon_start=1
                     /product="nuclear localization signal from the C. elegans 
                     EGL-13 transcription factor (Lyssenko et al., 2007)"
                     /label=EGL-13 NLS
                     /translation="SRRRKANPTKLSENAKKLAKEVEN"
     protein_bind    complement(3813..3912)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3929..3948)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3930..3948)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3953..3969)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3953..3969)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4071..4090)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4082..4891
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4149..4168)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      5038..5626
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5527..5546
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5780..5797
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(5956..5983)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(6075..6161)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     join(6210..6224,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(6224,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtctt aagctcgggc cccaaataat gattttattt tgactgatag
       61 tgacctgttc gttgcaacaa attgatgagc aatgcttttt tataatgcca actttgtaca
      121 aaaaagcagg ctcgggatcc ccaaagaaga agcgtaaggt gtccaaggta acacttagtt
      181 tttgttgctc tgtttaaaaa ttaattttaa gggagaggag gacaacatgg cctccctccc
      241 agccacccac gagctccaca tcttcggctc catcaacgga gtcgacttcg acatggtcgg
      301 acaaggaacc ggaaacccaa acgacggata cgaggagctc aacctcaagt ccaccaaggt
      361 aagttttctg ttttatttac ttgttttgag aattgggatt tttagctgaa aaattggatt
      421 ttaagatgaa aaattgaaat tttcaactaa aaatttcaat ttttagctga aaagttaaat
      481 ttttagctaa agagtgggat ttttagttga aaaatcgaga tttctagcta aaaagtatga
      541 tttttagctg aaagattgga attttaagca gaaaaaattg tatttttagc tgataaatca
      601 aattttaagc ggaaaaattt gaatttttag ctaaaaaatt ccattttcaa ctaaaaatcg
      661 tgatttttag ctttaaaaat tggattttta gctgaaaaat cggattttta gctgaaaatt
      721 gaaatttttt agttaaaaag tgagattttt taacagaaaa gtgagatttt tagttaaaaa
      781 ataatatttt tagctaaaaa atacaatttt caactaaaga tcgtgatttt tagctttaaa
      841 aattggattt ttagctaaaa aatcggattt ttagctgaaa attgaaattt tttagttaaa
      901 aagtgggatt ttaagatgaa aagtgagatt ttttagctga aaagtgagat tttcagctaa
      961 aaaatacaat tttcaactga aaaattggga tttttagctt taaaaattgg attttcagct
     1021 ttaaaaattg gatttttagc tgaaaagtga gatttttagt tgaaaaattg aaatgtttag
     1081 ctgaaaaatt ggaattttta agtaaaaaag tgggatttta agatgaaaag tgagatgttt
     1141 ttagctgaaa agtgagattt ttagctaaaa aatacaattt tcaactgaaa aattgggatt
     1201 tttagctcaa aagtgggacg aaagacgaaa aaaaaaatcg attttttacc aaaaattaga
     1261 ttttcagggt gacctccaat tctccccatg gatactcgtc ccacacatcg gatacggatt
     1321 ccaccaatac ctcccatacc cagacggaat gtccccattc caagccgcca tggtcgacgg
     1381 gtccggatac caagtccacc gtaccatgca attcgaggac ggagcctccc tcaccgtcaa
     1441 ctaccgttac acctacgagg gatcgcacat caaggtaagt tatgtacgcg atttctcggc
     1501 gaaaaaatgg acttttttgc tcaaaaactg aatttttcag ctttttttgc actgaaaatg
     1561 ctgaaaattg gtttttttgc tgaaaattca ccgaaaaaag ctgaaaatat tgcagaaatc
     1621 gacgaaaaat gccttttttg ctgaaatttc agccagaaaa ttgttcagaa tcgcaatttc
     1681 ctgccaaaat cgacgttttt cggcgaaaat tggtagattt tgaaccgaaa attgtgaaaa
     1741 atggactttt ttgctcaaaa acagactttt tgagcttttt tcacactgaa aatggtggaa
     1801 aattggattt tccagcagaa aattcggaaa ttttcaattt ttggcggaaa tttgacgaaa
     1861 aatatatgaa aaacaccgat ttttgtctga aaaagttcaa aagccgaaaa attggccaaa
     1921 aatctccaaa atcggacttt tctggcgttt ttcgtggaaa aattgaaatt tttttgaaaa
     1981 attgagcaaa aatcgccgaa aaaccgctaa aaaaactgcc gaaattggca attttgagct
     2041 taaaaaaact tgaaaaatgc tcaaaattac ccaattttca ccggaaattg agctaaaatc
     2101 gtcgaaaaag actgaatttt caatttttag ctgaaaatct gcctattttc ggctaaaatt
     2161 gctttaaaaa cattaaaatt caaatttttt ggctaaaaat actcagattg cccttttttt
     2221 tctccaaaaa tgacaaaaaa aaaatcaatc agaaaaatgc gatttttttt acaaaaattg
     2281 gcttcaaaac gtctaaattc tccctaattt ttgctgctga aaattgtgca aaaatcaccg
     2341 aaaaatgacg attttaggct gaaaatcttc aaaaaaagct caaaattccc cacttttcac
     2401 cggaaaaaga cgaaaaaagc tccaaattta tcgattaaca ttttcaggga gaggcccaag
     2461 tcaagggaac cggattccca gccgacggac cagtcatgac caactccctc accgccgccg
     2521 actggtgccg ttccaagaag acctacccaa acgacaagac catcatctcc accttcaagt
     2581 ggtcctacac caccggaaac ggaaagcgtt accgttccac cgcccgtacc acctacacct
     2641 tcgccaagcc aatggccgcc aactacctca aggtaagttc ttttttttgc tcgtttttga
     2701 gtgaaaaatt gatggaattc acaaaaaatg gccaaagaat cctcaaaatt ggccgaaaat
     2761 tctgaaaaat gacctaaaaa tcaccgaaaa acggccaaaa attatctgaa aatcgccgaa
     2821 aatctcaaat tttgaggaaa ttcccgctaa aaattaataa aaatcaccga aaaaaggcca
     2881 aaaattacct aataaaggcc gaaaatcgcc taaaaaggcc aaaaattctg aaaaatggcc
     2941 aaatttcaat ttttcacaca caaaaaatcc ctgaattttc tatttttact cgaaaaactg
     3001 tggatttacg ggggtaaaaa ttcagttttt agtgctattt cacaagaaaa acacagcatt
     3061 ttcgacaaaa actccgattt ttaagccaaa aatttgaggt ttttagtttt ttacgtgaaa
     3121 acctgaactt tttgccaaaa aaacactttt ttttagacta aaaatcaatt ttcgatcaaa
     3181 aatcactaat tttgcctgaa aaaatataga tttttccact ttttcactga aaaaaaactg
     3241 gattttcact agaaaatcaa gttgttttta gaccaaaaat tttatttttc cgctgaaaat
     3301 tatcgttttt cagtcaattt tcagctaaaa atccctgaaa ttctggattt tcagccaaaa
     3361 aactcgaaat tttccaaaaa aaccccgttt aaaaatgagt ttagactgaa aattccgatt
     3421 tttgagccaa aaatttgatt tttaatcgaa aaaaattgga ttttcaacca aaaaatcagt
     3481 ttttcagtga tttttagagt aaaaatcact gattttaaca caaaattcca gtttttcgtg
     3541 atttttcact agaaaatccc gatttttccc caaaaaatcc atttttccga ctaaaaatcc
     3601 ccaaagtaat tttcagaacc aaccaatgta cgtcttccgt aagaccgagc tcaagcactc
     3661 caagaccgag ctcaacttca aggagtggca aaaggccttc accgacgtca tgggaatgga
     3721 cgagctctac aagtcccgtc gtcgtaaggc caacccaacc aagctctccg agaacgccaa
     3781 gaagctcgcc aaggaggtcg agaacggatc caacccagct ttcttgtaca aagttggcat
     3841 tataagaaag cattgcttat caatttgttg caacgaacag gtcactatca gtcaaaataa
     3901 aatcattatt tgccatccag ctgatatccc ctatagtgag tcgtattaca tggtcatagc
     3961 tgtttcctgg cagctctggc ccgtgtctca aaatctctga tgttacattg cacaagataa
     4021 aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt
     4081 tatgagccat attcaacggg aaacgtcgag gccgcgatta aattccaaca tggatgctga
     4141 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg
     4201 cttgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc
     4261 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc
     4321 gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc
     4381 cggaaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga
     4441 tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa
     4501 cagcgatcgc gtatttcgcc tcgctcaggc gcaatcacga atgaataacg gtttggttga
     4561 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat
     4621 gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga
     4681 taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat
     4741 cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc
     4801 attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca
     4861 gtttcatttg atgctcgatg agtttttcta atcagaattg gttaattggt tgtaacactg
     4921 gcagagcatt acgctgactt gacgggacgg cgcaagctca tgaccaaaat cccttaacgt
     4981 gagttacgcg tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     5041 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     5101 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     5161 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     5221 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     5281 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     5341 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     5401 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     5461 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     5521 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     5581 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     5641 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     5701 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     5761 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
     5821 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     5881 cgactggaaa gcgggcagtg agcgcaacgc aattaatacg cgtaccgcta gccaggaaga
     5941 gtttgtagaa acgcaaaaag gccatccgtc aggatggcct tctgcttagt ttgatgcctg
     6001 gcagtttatg gcgggcgtcc tgcccgccac cctccgggcc gttgcttcac aacgttcaaa
     6061 tccgctcccg gcggatttgt cctactcagg agagcgttca ccgacaaaca acagataaaa
     6121 cgaaaggccc agtcttccga ctgagccttt cgttttattt gatgcctggc agttccctac
     6181 tctcgcgtta acgctagcat ggatgttttc ccagtcacga cgtt
//
