LOCUS       sequence-382094-        9070 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual-selection cassette plasmid for knocking-in AID-mTagBFP2 into 
            the C. elegans genome.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9070)
  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 9070)
  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-mTagBFP2-loxP_myo2_neoR-loxP
FEATURES             Location/Qualifiers
     source          1..9070
                     /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(1443..1476)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    2497..2521
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2554..2582)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3330..3350
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(4412..4428)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             5250..6044
                     /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(5304..5323)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5914..5933
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    complement(6774..6807)
                     /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(6849..6865)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6849..6865)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6862..6884)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6873..6889
                     /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(6897..6903,6904..6921,6922..6927))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6942..6963
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7080..7097)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7251..7839)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7331..7350)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8010..8801,8802..8870))
                     /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     8633..8652
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8871..8975)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     9043..9061
                     /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 aaggtctcca agggagagga gctcatcaag gagaacatgc
      601 acatgaagct ctacatggag ggaaccgtcg acaaccacca cttcaagtgc acctccgagg
      661 gagagggaaa gccatacgag ggaacccaaa ccatgcgtat caaggtcgtc gagggaggac
      721 cactcccatt cgccttcgac atcctcgcca cctccttcct ctacggatcc aaggtaagtt
      781 taaacatata tatactaact aaccctgatt atttaaattt tcagaccttc atcaaccaca
      841 cccaaggaat cccagacttc ttcaagcaat ccttcccaga gggattcacc tgggagcgtg
      901 tcaccaccta cgaggacgga ggagtcctca ccgccaccca agacacctcc ctccaagacg
      961 gatgcctcat ctacaacgtc aaggtaagtt taaacagttc ggtactaact aaccatacat
     1021 atttaaattt tcagatccgt ggagtcaact tcacctccaa cggaccagtc atgcaaaaga
     1081 agaccctcgg atgggaggcc ttcaccgaga ccctctaccc agccgatgga ggactcgagg
     1141 gacgtaacga catggccctc aagctcgtcg gaggatccca cctcatcgcc aacgccaagg
     1201 taagtttaaa catgatttta ctaactaact aatctgattt aaattttcag accacctacc
     1261 gttccaagaa gccagccaag aacctcaaga tgccaggagt ctactacgtc gactaccgtc
     1321 tcgagcgtat caaggaggcc aacaacgaga cctacgtcga gcaacacgag gtcgccgtcg
     1381 cccgttactg cgacctccca tccaagctcg gacacaagct caactaacgc atcaggcggc
     1441 ctataacttc gtatagcata cattatacga agttattacg tagagctcgg tacctcgtac
     1501 gtaatacgac tcactagtca ttttatatct gagtagtacc tttgctttaa atgtccataa
     1561 aactaatttt ataatcaata aaacaacgtt tgtaaatcaa ctgagtttac aagtagagac
     1621 attgagggat actttcacta tgctaaagtg ataatcgacc aaataataac ttcactttgg
     1681 tatttattcc tgtcttataa atgttatgta tgaattaaat tcatatgcat atggctcact
     1741 ctgacaattt ttaataatct tccagatcaa tattgactac cgatgcgggt ggtcttttgc
     1801 tttgaattct gctgaacttt acaccccgaa cagcaatgtg tgcttcagcc taaaaaaaag
     1861 taagtgtgtt aatcagtgcc cccgattctt cattttttgc ccctctctcc cgtttcgtcg
     1921 gcaaaagaag agaaaataaa gataagtctc aagataggtt ggtaatcgct aaagtggttg
     1981 tgtggataag agtagcaaaa tggcaggaag agcactttgc gcgcacacac tgtactcatt
     2041 gttctggata aaattctctc gttgtttgcc gtcggatgtc tgcctctctg cattgagccg
     2101 gcttcttcac tatctttagt taacctaaaa tgccgtttct tttctcgtat ccccactatc
     2161 ccgttgaggt tctctgctct cttcgctccc taccgccagc gagcaactat ccgtgggggc
     2221 gccttgctcg gaagatgggg gggaagaaag aagatttttg ctatttgcac ttgagaaaga
     2281 gacttttcct gcgtcgatgg tcagagaaca gtgtgcagac acttttcagc tacctagaat
     2341 tacaattgga tatccccgcc tcccaatcca cccacccagg gaaaaagaag ggctcgccga
     2401 aaatcaaagt tatctccagg ctcgcgcatc ccaccgagcg gttgacttct ctccaccact
     2461 tttcatttta accctcgatc gtcagacaca gaaatgacaa gtttgtacaa aaaagcaggc
     2521 ttaatgagta aaggagaaga acttttcact ggagttgtcc caattcttgt tgaattagat
     2581 ggtgatgtta atgggcacaa attttctgtc agtggagagg gtgaaggtga tgcaacatac
     2641 ggaaaactta cccttaaatt tatttgcact actggaaaac tacctgttcc atgggtaagt
     2701 ttaaacatat atatactaac taaccctgat tatttaaatt ttcagccaac acttgtcact
     2761 actttctgtt atggtgttca atgcttctcg agatacccag atcatatgaa acggcatgac
     2821 tttttcaaga gtgccatgcc cgaaggttat gtacaggaaa gaactatatt tttcaaagat
     2881 gacgggaact acaagacacg taagtttaaa cagttcggta ctaactaacc atacatattt
     2941 aaattttcag gtgctgaagt caagtttgaa ggtgataccc ttgttaatag aatcgagtta
     3001 aaaggtattg attttaaaga agatggaaac attcttggac acaaattgga atacaactat
     3061 aactcacaca atgtatacat catggcagac aaacaaaaga atggaatcaa agttgtaagt
     3121 ttaaacatga ttttactaac taactaatct gatttaaatt ttcagaactt caaaattaga
     3181 cacaacattg aagatggaag cgttcaacta gcagaccatt atcaacaaaa tactccaatt
     3241 ggcgatggcc ctgtcctttt accagacaac cattacctgt ccacacaatc tgccctttcg
     3301 aaagatccca acgaaaagag agaccacatg gtccttcttg agtttgtaac agctgctggg
     3361 attacacatg gcatggatga actatacaaa tagcattcgt agaattccaa ctgagcgccg
     3421 gtcgctacca ttaccaactt gtctggtgtc aaaaataata ggggccgctg tcatcagagt
     3481 aagtttaaac tgagttctac taactaacga gtaatattta aattttcagc atctcgcgcc
     3541 cgtgcctctg acttctaagt ccaattactc ttcaacatcc ctacatgctc tttctccctg
     3601 tgctcccacc ccctattttt gttattatca aaaaacttct cttaatttct ttgtttttta
     3661 gcttctttta agtcacctct aacaatgaaa ttgtgtagat tcaaaaatag aattaattcg
     3721 taataaaaag tcgaaaaaaa ttgtgctccc tccccccatt aataataatt ctatcccaaa
     3781 atctacacaa tgttctgtgt acacttctta tgttttttac ttctgataaa tttttttgaa
     3841 acatcataga aaaaaccgca cacaaaatac cttatcatat gttacgtttc agtttatgac
     3901 cgcaattttt atttcttcgc acgtctgggc ctctcatgac gtcaaatcat gctcatcgtg
     3961 aaaaagtttt ggagtatttt tggaattttt caatcaagtg aaagtttatg aaattaattt
     4021 tcccgctttt gctttttggg gtttccccta ttgtttgtca agatttcgag gacggcgttt
     4081 ttcttgctaa aatcacaagt attgatgagc acgatgcaag aaagatcgga agaaggtttg
     4141 ggtttgaggc tcagtggaag gtgagtagaa gttgataatt tgaaagtgga gtagtgtcta
     4201 tggggttttt gccttaaatg acagaataca ttcccaatat accaaacata actgtttaga
     4261 tcttcgaatg catcgcgcgc accgtacgtc tcgaggaatt cctgcaggat atctggatcc
     4321 acgaagcttc ccatggtgac gtcaccggtt ctagatacct aggtgagctc tggtaccctc
     4381 tagtcaaggc cttaagtgag tcgtattacg gactggccgt cgttttacag gccggatggg
     4441 ccatggcacc ttaacatttc taaattttag tttaataaaa aaattttaaa ataatacgag
     4501 ttttgagact aactttcaat aaaaaccaaa aaattgcttc aaaaaagagc tcgcgccgat
     4561 ttacgataaa cacagcgcgc tacgacactc cgaaaaccgc gccgcaccat tacattctcc
     4621 gccccaccgc acggtttgcg gaaggcgatg aaattgctgg gcctaaaatt ttaacccttt
     4681 ttttcccgtt tttcaactgt tttcccgatt ttttcgatat ttttttcagg tttttagtta
     4741 gaaaagtata taaaaattgc attatttgta gttttcgctc gtaatttgtg atagaaaacg
     4801 gccaatttgc ctagatttgt gattctacca agtggaattt ttgggaaaat atcaatattc
     4861 cacgattttt attcaaatca taacgtttcg aaattatcag cttaaataaa actaaaataa
     4921 ataataaaat actaaaaacc ctgaatttct cggttctcgg ttcagcttga gttcgttcta
     4981 tacacgttat cgatttttgt catttcgttt aatcatgggt tttgattcat tttaaatatg
     5041 aaatttgatt tattcctcgt ttcgaagttg gtttgtcttg tccggccggt aaacaaatgt
     5101 tattaaaaaa aataataaat ttcgaaattt ttcgtaatgt tccgcaatcg tgtttttccg
     5161 tttatctcgt cttcccttcc ttcaaatata ctatttattt tctcaattca catattttca
     5221 gccagccctg ctcaacaagt ggaataaaaa tgattgaaca agatggattg cacgcaggtt
     5281 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     5341 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     5401 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
     5461 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     5521 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     5581 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     5641 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     5701 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcacca gccgaactgt
     5761 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     5821 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     5881 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     5941 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     6001 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagtccaa ttactcttca
     6061 acatccctac atgctctttc tccctgtgct cccaccccct atttttgtta ttatcaaaaa
     6121 acttctctta atttctttgt tttttagctt cttttaagcc acctctaaca atgaaattgt
     6181 gtagattcaa aaatagaatt aattcgtaat aaaaagtcga aaaaaattgt gctccctccc
     6241 cccattaata ataattctat cccaaaatct acacaatgtt ctgtgtacac ttcttatgtt
     6301 ttttacttct gataaatttt tttgaaacat catagaaaaa accgcacaca aaatacctta
     6361 tcatatgtta cgtttcagtt tatgaccgca atttttattt cttcgcacgt ctgggcctct
     6421 catgacgtca aatcatgctc atcgtgaaaa agttttggag tatttttgga atttttcaat
     6481 caagtgaaag tttatgaaat taattttcct gcttttgctt tttggggttt cccctattgt
     6541 ttgtcaagat ttcgaggacg gcgtttttct tgctaaaatc acaagtattg atgagcacga
     6601 tgcaagaaag atcggaagaa ggtttgggtt tgaggctcag tggaaggtga gtagaagttg
     6661 ataatttgaa agtggagtag tgtctatggg gtttttgcct taaatgacag aatacattcc
     6721 caatatacca aacataactg tttctcgagg tcgacaggcc tgcatgcgtc gacataactt
     6781 cgtatagcat acattatacg aagttatagt tgcaggacca ctgcggccgc aagcttggcg
     6841 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     6901 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     6961 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     7021 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     7081 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     7141 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     7201 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     7261 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     7321 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     7381 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     7441 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     7501 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     7561 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     7621 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     7681 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     7741 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     7801 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     7861 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     7921 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     7981 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     8041 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     8101 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     8161 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     8221 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     8281 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     8341 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     8401 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     8461 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     8521 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     8581 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     8641 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     8701 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     8761 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     8821 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     8881 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     8941 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     9001 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     9061 ccctttcgtc
//
