LOCUS       sequence-426508-        6037 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3mAID for N-terminal tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6037)
  AUTHORS   Hatoyama Y, Islam M, Bond AG, Hayashi KI, Ciulli A, Kanemaki MT
  TITLE     Combination of AID2 and BromoTag expands the utility of degron-based
            protein knockdowns.
  JOURNAL   EMBO Rep. 2024 Sep;25(9):4062-4077. doi: 10.1038/s44319-024-00224-4.
            Epub 2024 Aug 23.
  PUBMED    39179892
REFERENCE   2  (bases 1 to 6037)
  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"; doi: 
            "10.1038/s44319-024-00224-4"; journalName: "EMBO Rep"; date: 
            "2024-09"; volume: "25"; issue: "9"; pages: "4062-4077"
COMMENT     Sequence Label: pMK485 (3mAID-Hygro)
FEATURES             Location/Qualifiers
     source          1..6037
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      4..459
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(91..110)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     301..322
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             702..905
                     /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             930..1133
                     /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             1158..1361
                     /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"
     polyA_signal    1379..1500
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1416..1435)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1470..1489
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    1531..1564
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1625..2124
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1660..1683)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2057..2076
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2185..3204
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             2185..3204
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     protein_bind    3767..3800
                     /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(3849..3869)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3849..3867)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3888..3904)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3888..3904)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3901..3923)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3912..3928
                     /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(3936..3942,3943..3960,3961..3966))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3981..4002
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4119..4136)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4290..4878)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4370..4389)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5049..5840,5841..5909))
                     /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     5672..5691
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5910..6014)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
       61 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
      121 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
      181 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
      241 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
      301 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
      361 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
      421 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctccc
      661 cgggatccgg tgctggggca ggcgctggtg ctggcgctgg taaagagaaa agcgcatgtc
      721 ctaaagatcc tgcaaagcct ccagccaaag cacaggtggt tggatggcct cccgttagaa
      781 gttataggaa gaacgttatg gtaagctgcc aaaagtctag cggtgggccc gaagcggcgg
      841 cttttgtaaa agtgagcatg gatggagccc catatttgag gaaaattgat ctcagaatgt
      901 ataagggggc gggtgctggt gccggtgcaa aggaaaagtc tgcgtgtccc aaagaccctg
      961 caaaacctcc ggcaaaggca caggtcgtgg gttggccgcc agtgcgatcc tatcggaaaa
     1021 acgtaatggt tagctgccag aagtcttcag ggggtcccga agctgcggcg tttgtgaaag
     1081 taagtatgga tggtgctccg tatctccgaa aaatcgacct cagaatgtat aaaggtgcag
     1141 gtgcaggagc gggggcgaaa gagaagtccg cctgtccaaa agatcctgct aagccaccag
     1201 ccaaggcaca agtggtcggt tggccaccag tgagaagtta tcgaaagaac gtaatggtgt
     1261 catgtcaaaa gtcaagcgga ggacctgaag cagctgcctt cgtcaaggta tcaatggacg
     1321 gcgcgcctta tctgagaaaa atcgatttga gaatgtacaa ataacaattg ttgttgttaa
     1381 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     1441 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     1501 acgcgtcgat catattcaat aacccttaat ataacttcgt ataatgtatg ctatacgaag
     1561 ttattaggtc tgaagaggag tttacgtcca gccaagctta ggatctcgac ctcgaaattc
     1621 taccgggtag gggaggcgct tttcccaagg cagtctggag catgcgcttt agcagccccg
     1681 ctgggcactt ggcgctacac aagtggcctc tggcctcgca cacattccac atccaccggt
     1741 aggcgccaac cgactccgtt ctttggtggc cccttcgcgc caccttctac tcctccccta
     1801 gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta
     1861 gcacgtctca ctagtctcgt gcagatggac agcaccgctg agcaatggaa gcgggtaggc
     1921 ctttggggca gcggccaata gcagctttgc tccttcgctt tctgggctca gaggctggga
     1981 aggggtgggt ccgggggcgg gctcaggggc gggctcaggg gcggggcggg cgcccgaagg
     2041 tcctccggag gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc
     2101 tcttcctcat ctccgggcct ttcgacctgc atccatctag atctcgagca gctgaagctt
     2161 accgctagca tggatagatc cggaaagcct gaactcaccg cgacgtctgt cgagaagttt
     2221 ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct
     2281 cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc
     2341 gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt
     2401 ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat ctcccgccgt
     2461 gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg
     2521 gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc
     2581 ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt
     2641 gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc
     2701 gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc
     2761 gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc
     2821 attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc
     2881 tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg
     2941 gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc
     3001 tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac
     3061 gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg
     3121 gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc
     3181 actcgtccga gggcaaagga ataggctagc cgcccgcccc acgacccgca gcgcccgacc
     3241 gaaaggagcg cacgacccca tgcatcgatg atatcagatc cccgggatgc agaaattgat
     3301 gatctattaa acaataaaga tgtccactaa aatggaagtt tttcctgtca tactttgtta
     3361 agaagggtga gaacagagta cctacatttt gaatggaagg attggagcta cgggggtggg
     3421 ggtggggtgg gattagataa atgcctgctc tttactgaag gctctttact attgctttat
     3481 gataatgttt catagttgga tatcataatt taaacaagca aaaccaaatt aagggccagc
     3541 tcattcctcc cactcatgat ctatagatct atagatctct cgtgggatca ttgtttttct
     3601 cttgattccc actttgtggt tctaagtact gtggtttcca aatgtgtcag tttcatagcc
     3661 tgaagaacga gatcagcagc ctctgttcca catacacttc attctcagta ttgttttgcc
     3721 aagttctaat tccatcagaa gctggtcgag atccggaacc cttaatataa cttcgtataa
     3781 tgtatgctat acgaagttat taggtccctc gaagaggttc actaggatcc ggtacccagc
     3841 ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt
     3901 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     3961 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     4021 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     4081 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4141 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4201 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4261 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4321 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4381 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4441 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4501 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4561 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4621 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4681 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     4741 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4801 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4861 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4921 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4981 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     5041 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     5101 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     5161 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     5221 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     5281 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     5341 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     5401 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     5461 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     5521 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     5581 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     5641 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     5701 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     5761 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     5821 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     5881 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     5941 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     6001 ataggggttc cgcgcacatt tccccgaaaa gtgccac
//
