LOCUS       sequence-382109-        9058 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual-selection cassette plasmid for knocking-in AID-BFP into the C. 
            elegans genome.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9058)
  AUTHORS   Kurashina M, Mizumoto K
  TITLE     Targeting endogenous proteins for spatial and temporal knockdown 
            using auxin-inducible degron in Caenorhabditis elegans.
  JOURNAL   STAR Protoc. 2023 Jan 13;4(1):102028. doi: 
            10.1016/j.xpro.2022.102028.
  PUBMED    36640369
REFERENCE   2  (bases 1 to 9058)
  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: "STAR 
            Protoc."; date: "2023-01-13"; volume: "4(1):102028. doi"; pages: " 
            10.1016/j.xpro.2022.102028"
COMMENT     Sequence Label: AID-BFP-loxP_myo2_neoR
FEATURES             Location/Qualifiers
     source          1..9058
                     /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     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             432..563
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Morawska and Ulrich, 
                     2013)"
                     /label=AID*
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="PKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEAAAFVK"
     protein_bind    complement(1431..1464)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    2485..2509
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2542..2570)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3318..3338
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(4400..4416)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             5238..6032
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5292..5311)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5902..5921
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    complement(6762..6795)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(6837..6853)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6837..6853)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6850..6872)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6861..6877
                     /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(6885..6891,6892..6909,6910..6915))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6930..6951
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7068..7085)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7239..7827)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7319..7338)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7998..8789,8790..8858))
                     /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     8621..8640
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8859..8963)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     9031..9049
                     /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 cccgcgggga gccggagccg
      421 gagccggagc ccctaaagat ccagccaaac ctccggccaa ggcacaagtt gtgggatggc
      481 caccggtgag atcataccgg aagaacgtga tggtttcctg ccaaaaatca agcggtggcc
      541 cggaggcggc ggcgttcgtg aagtcagagc ttattaagga gaatatgcat atgaaactgt
      601 acatggaagg aaccgtggac aaccatcact ttaagtgcac ttcggagggg gaaggcaaac
      661 cgtacgaggg cacccagacc atgcgaatca aggtggtcga gggaggccca ctgcccttcg
      721 ccttcgacat tctggtaagt ttaaacatat atatactaac taaccctgat tatttaaatt
      781 ttcaggctac tagcttcctg tacggcagca agacttttat taatcacacc cagggcatcc
      841 cggacttctt caaacagtcg ttccctgagg gcttcacatg ggagcgtgtg accacatacg
      901 aagacggtgg cgttctcacc gctacccagg acaccagcct ccaggacggt tgcctcatat
      961 ataatgtggt aagtttaaac agttcggtac taactaacca tacatattta aattttcaga
     1021 agatccgggg agtaaacttc acatccaatg gtccggttat gcagaagaaa acactcggct
     1081 gggaggcctt caccgagacg ctgtatccgg ctgacggagg cctggaaggt cgaaacgaca
     1141 tggccctgaa gctcgtcggc ggtagccatc tcatcgcaaa catcaagacc acatatagat
     1201 ccaagaaacc ggtaagttta aacatgattt tactaactaa ctaatctgat ttaaattttc
     1261 aggctaagaa ccttaaaatg ccgggagtct actatgttga ctacagactg gaacggatca
     1321 aggaggccaa caacgagacc tacgtcgagc agcacgaggt tgcagttgcc agatactgcg
     1381 acctcccgag caaactgggt cacaagctta attagcgcat caggcggcct ataacttcgt
     1441 atagcataca ttatacgaag ttattacgta gagctcggta cctcgtacgt aatacgactc
     1501 actagtcatt ttatatctga gtagtacctt tgctttaaat gtccataaaa ctaattttat
     1561 aatcaataaa acaacgtttg taaatcaact gagtttacaa gtagagacat tgagggatac
     1621 tttcactatg ctaaagtgat aatcgaccaa ataataactt cactttggta tttattcctg
     1681 tcttataaat gttatgtatg aattaaattc atatgcatat ggctcactct gacaattttt
     1741 aataatcttc cagatcaata ttgactaccg atgcgggtgg tcttttgctt tgaattctgc
     1801 tgaactttac accccgaaca gcaatgtgtg cttcagccta aaaaaaagta agtgtgttaa
     1861 tcagtgcccc cgattcttca ttttttgccc ctctctcccg tttcgtcggc aaaagaagag
     1921 aaaataaaga taagtctcaa gataggttgg taatcgctaa agtggttgtg tggataagag
     1981 tagcaaaatg gcaggaagag cactttgcgc gcacacactg tactcattgt tctggataaa
     2041 attctctcgt tgtttgccgt cggatgtctg cctctctgca ttgagccggc ttcttcacta
     2101 tctttagtta acctaaaatg ccgtttcttt tctcgtatcc ccactatccc gttgaggttc
     2161 tctgctctct tcgctcccta ccgccagcga gcaactatcc gtgggggcgc cttgctcgga
     2221 agatgggggg gaagaaagaa gatttttgct atttgcactt gagaaagaga cttttcctgc
     2281 gtcgatggtc agagaacagt gtgcagacac ttttcagcta cctagaatta caattggata
     2341 tccccgcctc ccaatccacc cacccaggga aaaagaaggg ctcgccgaaa atcaaagtta
     2401 tctccaggct cgcgcatccc accgagcggt tgacttctct ccaccacttt tcattttaac
     2461 cctcgatcgt cagacacaga aatgacaagt ttgtacaaaa aagcaggctt aatgagtaaa
     2521 ggagaagaac ttttcactgg agttgtccca attcttgttg aattagatgg tgatgttaat
     2581 gggcacaaat tttctgtcag tggagagggt gaaggtgatg caacatacgg aaaacttacc
     2641 cttaaattta tttgcactac tggaaaacta cctgttccat gggtaagttt aaacatatat
     2701 atactaacta accctgatta tttaaatttt cagccaacac ttgtcactac tttctgttat
     2761 ggtgttcaat gcttctcgag atacccagat catatgaaac ggcatgactt tttcaagagt
     2821 gccatgcccg aaggttatgt acaggaaaga actatatttt tcaaagatga cgggaactac
     2881 aagacacgta agtttaaaca gttcggtact aactaaccat acatatttaa attttcaggt
     2941 gctgaagtca agtttgaagg tgataccctt gttaatagaa tcgagttaaa aggtattgat
     3001 tttaaagaag atggaaacat tcttggacac aaattggaat acaactataa ctcacacaat
     3061 gtatacatca tggcagacaa acaaaagaat ggaatcaaag ttgtaagttt aaacatgatt
     3121 ttactaacta actaatctga tttaaatttt cagaacttca aaattagaca caacattgaa
     3181 gatggaagcg ttcaactagc agaccattat caacaaaata ctccaattgg cgatggccct
     3241 gtccttttac cagacaacca ttacctgtcc acacaatctg ccctttcgaa agatcccaac
     3301 gaaaagagag accacatggt ccttcttgag tttgtaacag ctgctgggat tacacatggc
     3361 atggatgaac tatacaaata gcattcgtag aattccaact gagcgccggt cgctaccatt
     3421 accaacttgt ctggtgtcaa aaataatagg ggccgctgtc atcagagtaa gtttaaactg
     3481 agttctacta actaacgagt aatatttaaa ttttcagcat ctcgcgcccg tgcctctgac
     3541 ttctaagtcc aattactctt caacatccct acatgctctt tctccctgtg ctcccacccc
     3601 ctatttttgt tattatcaaa aaacttctct taatttcttt gttttttagc ttcttttaag
     3661 tcacctctaa caatgaaatt gtgtagattc aaaaatagaa ttaattcgta ataaaaagtc
     3721 gaaaaaaatt gtgctccctc cccccattaa taataattct atcccaaaat ctacacaatg
     3781 ttctgtgtac acttcttatg ttttttactt ctgataaatt tttttgaaac atcatagaaa
     3841 aaaccgcaca caaaatacct tatcatatgt tacgtttcag tttatgaccg caatttttat
     3901 ttcttcgcac gtctgggcct ctcatgacgt caaatcatgc tcatcgtgaa aaagttttgg
     3961 agtatttttg gaatttttca atcaagtgaa agtttatgaa attaattttc ccgcttttgc
     4021 tttttggggt ttcccctatt gtttgtcaag atttcgagga cggcgttttt cttgctaaaa
     4081 tcacaagtat tgatgagcac gatgcaagaa agatcggaag aaggtttggg tttgaggctc
     4141 agtggaaggt gagtagaagt tgataatttg aaagtggagt agtgtctatg gggtttttgc
     4201 cttaaatgac agaatacatt cccaatatac caaacataac tgtttagatc ttcgaatgca
     4261 tcgcgcgcac cgtacgtctc gaggaattcc tgcaggatat ctggatccac gaagcttccc
     4321 atggtgacgt caccggttct agatacctag gtgagctctg gtaccctcta gtcaaggcct
     4381 taagtgagtc gtattacgga ctggccgtcg ttttacaggc cggatgggcc atggcacctt
     4441 aacatttcta aattttagtt taataaaaaa attttaaaat aatacgagtt ttgagactaa
     4501 ctttcaataa aaaccaaaaa attgcttcaa aaaagagctc gcgccgattt acgataaaca
     4561 cagcgcgcta cgacactccg aaaaccgcgc cgcaccatta cattctccgc cccaccgcac
     4621 ggtttgcgga aggcgatgaa attgctgggc ctaaaatttt aacccttttt ttcccgtttt
     4681 tcaactgttt tcccgatttt ttcgatattt ttttcaggtt tttagttaga aaagtatata
     4741 aaaattgcat tatttgtagt tttcgctcgt aatttgtgat agaaaacggc caatttgcct
     4801 agatttgtga ttctaccaag tggaattttt gggaaaatat caatattcca cgatttttat
     4861 tcaaatcata acgtttcgaa attatcagct taaataaaac taaaataaat aataaaatac
     4921 taaaaaccct gaatttctcg gttctcggtt cagcttgagt tcgttctata cacgttatcg
     4981 atttttgtca tttcgtttaa tcatgggttt tgattcattt taaatatgaa atttgattta
     5041 ttcctcgttt cgaagttggt ttgtcttgtc cggccggtaa acaaatgtta ttaaaaaaaa
     5101 taataaattt cgaaattttt cgtaatgttc cgcaatcgtg tttttccgtt tatctcgtct
     5161 tcccttcctt caaatatact atttattttc tcaattcaca tattttcagc cagccctgct
     5221 caacaagtgg aataaaaatg attgaacaag atggattgca cgcaggttct ccggccgctt
     5281 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     5341 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     5401 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     5461 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     5521 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     5581 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     5641 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     5701 aggatgatct ggacgaagag catcaggggc tcgcaccagc cgaactgttc gccaggctca
     5761 aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     5821 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     5881 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     5941 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     6001 ccttctatcg ccttcttgac gagttcttct gagtccaatt actcttcaac atccctacat
     6061 gctctttctc cctgtgctcc caccccctat ttttgttatt atcaaaaaac ttctcttaat
     6121 ttctttgttt tttagcttct tttaagccac ctctaacaat gaaattgtgt agattcaaaa
     6181 atagaattaa ttcgtaataa aaagtcgaaa aaaattgtgc tccctccccc cattaataat
     6241 aattctatcc caaaatctac acaatgttct gtgtacactt cttatgtttt ttacttctga
     6301 taaatttttt tgaaacatca tagaaaaaac cgcacacaaa ataccttatc atatgttacg
     6361 tttcagttta tgaccgcaat ttttatttct tcgcacgtct gggcctctca tgacgtcaaa
     6421 tcatgctcat cgtgaaaaag ttttggagta tttttggaat ttttcaatca agtgaaagtt
     6481 tatgaaatta attttcctgc ttttgctttt tggggtttcc cctattgttt gtcaagattt
     6541 cgaggacggc gtttttcttg ctaaaatcac aagtattgat gagcacgatg caagaaagat
     6601 cggaagaagg tttgggtttg aggctcagtg gaaggtgagt agaagttgat aatttgaaag
     6661 tggagtagtg tctatggggt ttttgcctta aatgacagaa tacattccca atataccaaa
     6721 cataactgtt tctcgaggtc gacaggcctg catgcgtcga cataacttcg tatagcatac
     6781 attatacgaa gttatagttg caggaccact gcggccgcaa gcttggcgta atcatggtca
     6841 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     6901 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     6961 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     7021 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     7081 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     7141 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     7201 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     7261 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     7321 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     7381 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     7441 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     7501 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     7561 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     7621 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     7681 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     7741 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     7801 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     7861 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     7921 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     7981 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     8041 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     8101 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     8161 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     8221 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     8281 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     8341 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     8401 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     8461 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     8521 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     8581 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     8641 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     8701 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     8761 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     8821 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     8881 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     8941 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     9001 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc
//
