LOCUS       sequence-234566-        4795 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-terminal tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4795)
  AUTHORS   Yesbolatova A, Natsume T, Hayashi KI, Kanemaki MT
  TITLE     Generation of conditional auxin-inducible degron (AID) cells and 
            tight control of degron-fused proteins using the degradation 
            inhibitor auxinole.
  JOURNAL   Methods. 2019 Apr 23. pii: S1046-2023(18)30331-1. doi: 
            10.1016/j.ymeth.2019.04.010.
  PUBMED    31026591
REFERENCE   2  (bases 1 to 4795)
  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: "Methods. 
            2019 Apr 23. pii: S1046-2023(18)30331-1. doi: 
            10.1016/j.ymeth.2019.04.010."
COMMENT     Sequence Label: pMK401 (Hygro-P2A-mAID-Nluc)
FEATURES             Location/Qualifiers
     source          1..4795
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     96..118
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     110..127
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     111..127
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     137..156
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        137..155
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     180..196
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     181..197
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             218..1240
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             218..1240
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             1253..1309
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1316..1519
                     /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             1526..2038
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     primer_bind     complement(2064..2080)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2064..2080)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2117..2137)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2117..2135)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2156..2172)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2156..2172)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2169..2191)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2180..2196
                     /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(2204..2210,2211..2228,2229..2234))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2249..2270
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2387..2404)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2558..3146)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2638..2657)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3317..4108,4109..4177))
                     /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     3940..3959
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4178..4282)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4309..4765
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4396..4415)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4606..4627
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc
       61 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
      121 ggccagtgag cgcgcgtaat acgactcact atagggcgaa ttgggtaccg ggccccccct
      181 cgaggtcgac ggtatcgata agcttgatat cgccaccatg aaaaagcctg aactcaccgc
      241 gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct
      301 ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct
      361 gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc
      421 atcggccgcg ctcccgattc cggaagtgct tgacattggg gaattcagcg agagcctgac
      481 ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact
      541 gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag
      601 ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg
      661 tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga
      721 caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg
      781 ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa
      841 tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga
      901 ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta
      961 cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt atatgctccg
     1021 cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg
     1081 ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca
     1141 aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag
     1201 tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa tcaggaagcg gagctactaa
     1261 cttcagcctg ctgaagcagg ctggagacgt ggaggagaac cctggaccta gatctaagga
     1321 gaagagtgct tgtcctaaag atccagccaa acctccggcc aaggcacaag ttgtgggatg
     1381 gccaccggtg agatcatacc ggaagaacgt gatggtttcc tgccaaaaat caagcggtgg
     1441 cccggaggcg gcggcgttcg tgaaggtatc aatggacgga gcaccgtact tgaggaaaat
     1501 cgatttgagg atgtataaag ctagcatggt cttcacactc gaagatttcg ttggggactg
     1561 gcgacagaca gccggctaca acctggacca agtccttgaa cagggaggtg tgtccagttt
     1621 gtttcagaat ctcggggtgt ccgtaactcc gatccaaagg attgtcctga gcggtgaaaa
     1681 tgggctgaag atcgacatcc atgtcatcat cccgtatgaa ggtctgagcg gcgaccaaat
     1741 gggccagatc gaaaaaattt ttaaggtggt gtaccctgtg gatgatcatc actttaaggt
     1801 gatcctgcac tatggcacac tggtaatcga cggggttacg ccgaacatga tcgactattt
     1861 cggacggccg tatgaaggca tcgccgtgtt cgacggcaaa aagatcactg taacagggac
     1921 cctgtggaac ggcaacaaaa ttatcgacga gcgcctgatc aaccccgacg gctccctgct
     1981 gttccgagta accatcaacg gagtgaccgg ctggcggctg tgcgaacgca ttctggcggg
     2041 cgctggtgca ggggccggcg ccggatccac tagttctaga gcggccgcca ccgcggtgga
     2101 gctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat
     2161 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     2221 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     2281 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     2341 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     2401 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     2461 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     2521 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     2581 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     2641 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     2701 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     2761 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     2821 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     2881 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     2941 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     3001 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     3061 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     3121 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     3181 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     3241 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     3301 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     3361 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     3421 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     3481 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     3541 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     3601 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     3661 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     3721 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     3781 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     3841 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     3901 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     3961 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     4021 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     4081 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     4141 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     4201 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     4261 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gcgccctgta
     4321 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     4381 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     4441 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     4501 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     4561 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     4621 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     4681 cgatttcggc ctattggtta aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     4741 aacaaaatat taacgcttac aatttccatt cgccattcag gctgcgcaac tgttg
//
