LOCUS       sequence-426507-        5809 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  2mAID for N-terminal tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5809)
  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 5809)
  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: pMK484 (2mAID-Hygro)
FEATURES             Location/Qualifiers
     source          1..5809
                     /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"
     polyA_signal    1151..1272
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1188..1207)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1242..1261
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    1303..1336
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1397..1896
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1432..1455)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1829..1848
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             1957..2976
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             1957..2976
                     /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    3539..3572
                     /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(3621..3641)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3621..3639)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3660..3676)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3660..3676)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3673..3695)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3684..3700
                     /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(3708..3714,3715..3732,3733..3738))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3753..3774
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3891..3908)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4062..4650)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4142..4161)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4821..5612,5613..5681))
                     /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     5444..5463
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5682..5786)
                     /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 aaataacaat
     1141 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     1201 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     1261 caatgtatct taacgcgtcg atcatattca ataaccctta atataacttc gtataatgta
     1321 tgctatacga agttattagg tctgaagagg agtttacgtc cagccaagct taggatctcg
     1381 acctcgaaat tctaccgggt aggggaggcg cttttcccaa ggcagtctgg agcatgcgct
     1441 ttagcagccc cgctgggcac ttggcgctac acaagtggcc tctggcctcg cacacattcc
     1501 acatccaccg gtaggcgcca accgactccg ttctttggtg gccccttcgc gccaccttct
     1561 actcctcccc tagtcaggaa gttccccccc gccccgcagc tcgcgtcgtg caggacgtga
     1621 caaatggaag tagcacgtct cactagtctc gtgcagatgg acagcaccgc tgagcaatgg
     1681 aagcgggtag gcctttgggg cagcggccaa tagcagcttt gctccttcgc tttctgggct
     1741 cagaggctgg gaaggggtgg gtccgggggc gggctcaggg gcgggctcag gggcggggcg
     1801 ggcgcccgaa ggtcctccgg aggcccggca ttctgcacgc ttcaaaagcg cacgtctgcc
     1861 gcgctgttct cctcttcctc atctccgggc ctttcgacct gcatccatct agatctcgag
     1921 cagctgaagc ttaccgctag catggataga tccggaaagc ctgaactcac cgcgacgtct
     1981 gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag
     2041 ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta
     2101 aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc
     2161 gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc
     2221 atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct
     2281 gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg
     2341 agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc
     2401 atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc
     2461 agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa
     2521 gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc
     2581 ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc
     2641 aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag
     2701 cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt
     2761 cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag
     2821 ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc
     2881 cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac
     2941 cgacgcccca gcactcgtcc gagggcaaag gaataggcta gccgcccgcc ccacgacccg
     3001 cagcgcccga ccgaaaggag cgcacgaccc catgcatcga tgatatcaga tccccgggat
     3061 gcagaaattg atgatctatt aaacaataaa gatgtccact aaaatggaag tttttcctgt
     3121 catactttgt taagaagggt gagaacagag tacctacatt ttgaatggaa ggattggagc
     3181 tacgggggtg ggggtggggt gggattagat aaatgcctgc tctttactga aggctcttta
     3241 ctattgcttt atgataatgt ttcatagttg gatatcataa tttaaacaag caaaaccaaa
     3301 ttaagggcca gctcattcct cccactcatg atctatagat ctatagatct ctcgtgggat
     3361 cattgttttt ctcttgattc ccactttgtg gttctaagta ctgtggtttc caaatgtgtc
     3421 agtttcatag cctgaagaac gagatcagca gcctctgttc cacatacact tcattctcag
     3481 tattgttttg ccaagttcta attccatcag aagctggtcg agatccggaa cccttaatat
     3541 aacttcgtat aatgtatgct atacgaagtt attaggtccc tcgaagaggt tcactaggat
     3601 ccggtaccca gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg
     3661 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     3721 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     3781 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     3841 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     3901 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     3961 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4021 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4081 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4141 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4201 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     4261 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4321 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4381 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4441 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     4501 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     4561 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     4621 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     4681 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     4741 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     4801 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     4861 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     4921 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     4981 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     5041 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     5101 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     5161 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     5221 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     5281 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     5341 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     5401 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     5461 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     5521 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     5581 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     5641 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     5701 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     5761 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccac
//
