LOCUS       sequence-345046-        5121 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid for tagging htz-1 with GFP::mAID.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5121)
  AUTHORS   Negishi T, Kitagawa S, Horii N, Tanaka Y, Haruta N, Sugimoto A, Sawa
            H, Hayashi KI, Harata M, Kanemaki MT
  TITLE     The auxin-inducible degron 2 (AID2) system enables controlled 
            protein knockdown during embryogenesis and development in 
            Caenorhabditis elegans.
  JOURNAL   Genetics. 2021 Dec 2. pii: 6448232. doi: 10.1093/genetics/iyab218.
  PUBMED    34865044
REFERENCE   2  (bases 1 to 5121)
  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: "Genetics. 
            2021 Dec 2. pii: 6448232. doi: 10.1093/genetics/iyab218."
COMMENT     Sequence Label: pGFPmAID_htz-1
FEATURES             Location/Qualifiers
     source          1..5121
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(168..756)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(248..267)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(927..1718,1719..1787))
                     /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     1550..1569
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1788..1892)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1919..2374
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2006..2025)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2216..2237
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2500..2522
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2514..2531
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2515..2531
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2538..2557
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2538..2556
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3121..3149)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3897..3917
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             complement(3989..4006)
                     /codon_start=1
                     /product="HQHQHQ metal affinity tag"
                     /label=HQ tag
                     /note="The HQ tag binds to nickel and other metal ions much
                     like the 6xHis tag, but may allow elution at lower 
                     concentrations of imidazole (Godat et al., 2008)."
                     /translation="HQHQHQ"
     CDS             4012..4215
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Kubota et al., 2013)"
                     /label=mini-AID
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEA
                     AAFVKVSMDGAPYLRKIDLRMYK"
     CDS             4287..4298
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        complement(4848..4866)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4887..4903)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4887..4903)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4900..4922)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4911..4927
                     /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(4935..4941,4942..4959,4960..4965))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4980..5001
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(join(5118..5121,1..14))
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
       61 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
      121 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
      181 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
      241 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
      301 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
      361 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
      421 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
      481 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
      541 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
      601 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
      661 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
      721 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
      781 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
      841 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
      901 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
      961 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     1021 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     1081 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     1141 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     1201 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     1261 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     1321 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     1381 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     1441 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     1501 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     1561 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     1621 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     1681 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     1741 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     1801 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     1861 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     1921 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     1981 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     2041 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     2101 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     2161 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     2221 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     2281 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     2341 gcgaatttta acaaaatatt aacgcttaca atttccattc gccattcagg ctgcgcaact
     2401 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
     2461 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
     2521 cgacggccag tgaattgtaa tacgactcac tatagggcga attggactag tgtccgaatt
     2581 agcatccgac gacatgtttc ttgaaatttg aatatttgtc accggaaccc ttcaagcttc
     2641 gattttaaaa ccgattttat tgaaaatacc ggttcgcgcg cttaaaaatt gcaaaccgag
     2701 agctccgccg taaatctaca cacccgaccg taccccacct cgttgcgaga cccttgcgta
     2761 tctaactagc tggcagcgtg ccaaaaagga ggagtctctc gtttgcgcgc gcttttgaga
     2821 gttgatttct tcgttcgaat gttttcctgt tcatttgtgc ataaattgca ctacgatttt
     2881 gcactaatgt ttttgtttgt attttgatcg aaaatcaagt aaaatgtatt ctttctggtg
     2941 tttctgaact gcttttaccc atttttcact atttcatcat tgtttctctg gagattcttc
     3001 gcttttatcg taattcaaca ttttgtggaa cgtttatcga tttttttaaa cattctgttc
     3061 ttgttttcag aagcttcaat ctagttcgca atgagtaaag gagaagaact tttcactgga
     3121 gttgtcccaa ttcttgttga attagatggt gatgttaatg ggcacaaatt ttctgtcagt
     3181 ggagagggtg aaggtgatgc aacatacgga aaacttaccc ttaaatttat ttgcactact
     3241 ggaaaactac ctgttccatg ggtaagttta aacatatata tactaactaa ccctgattat
     3301 ttaaattttc agccaacact tgtcactact ttctgttatg gtgttcaatg cttctcgaga
     3361 tacccagatc atatgaaacg gcatgacttt ttcaagagtg ccatgcccga aggttatgta
     3421 caggaaagaa ctatattttt caaagatgac gggaactaca agacacgtaa gtttaaacag
     3481 ttcggtacta actaaccata catatttaaa ttttcaggtg ctgaagtcaa gtttgaaggt
     3541 gatacccttg ttaatagaat cgagttaaaa ggtattgatt ttaaagaaga tggaaacatt
     3601 cttggacaca aattggaata caactataac tcacacaatg tatacatcat ggcagacaaa
     3661 caaaagaatg gaatcaaagt tgtaagttta aacatgattt tactaactaa ctaatctgat
     3721 ttaaattttc agaacttcaa aattagacac aacattgaag atggaagcgt tcaactagca
     3781 gaccattatc aacaaaatac tccaattggc gatggccctg tccttttacc agacaaccat
     3841 tacctgtcca cacaatctgc cctttcgaaa gatcccaacg aaaagagaga ccacatggtc
     3901 cttcttgagt ttgtaacagc tgctgggatt acacatggca tggatgaact atacaaatgt
     3961 ctagaggatc gggggcccgg tgccggtgct ggtgctggtg ctggtgccgg taaagaaaag
     4021 agtgcttgcc caaaagaccc tgctaaacct cctgccaagg ctcaagttgt aggatggcca
     4081 cctgttagat catacagaaa gaatgtcatg gtttcttgtc aaaagtcttc aggtggacca
     4141 gaagctgcag cctttgttaa agtatctatg gatggtgccc catatttgag aaagattgac
     4201 ttaaggatgt acaagggtgc tggagcaggc gccactagta tggctggagg aaaaggaaag
     4261 gccggaaagg actccggaaa gtccaaatcg aaggtcgtat cgagaagtgc tcgcgctgga
     4321 ctccaattcc ctgtcggacg tatccatcgt ttcttgaagc agagaaccac ttccagcgga
     4381 cgtgtcggag ctactgccgc agtatacagg tactgattcc atattcttga taataaaatt
     4441 ctaaaatttg ttttttcagc gctgccatcc tcgaatatct caccgccgaa gtcctcgagt
     4501 tggccggtaa cgcctccaag gatcttaagg tgaagcgtat caccccaaga catcttcatc
     4561 ttgctatcag aggagacgaa gagctcgaca ctctcatcaa ggccaccatc gccggaggag
     4621 gagtcatccc acacatccat cgttatttga tgaacaagaa gggagcccca gtcccaggaa
     4681 agccaggagc tccaggacaa ggaccacaat aaatgctgga catacctcaa atatctaaat
     4741 aatctaatat ttttctcaat ttcaccaaaa attaatctct catctatcac tccccaatag
     4801 gcgacgaatt aatttgatca ctgccactag ttaaccagct tttgttccct ttagtgaggg
     4861 ttaatttcga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     4921 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     4981 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     5041 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     5101 gggcgctctt ccgcttcctc g
//
