LOCUS       sequence-237883-        6195 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-terminal tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6195)
  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 6195)
  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: pMK408 (mAID-Nluc Hygro)
FEATURES             Location/Qualifiers
     source          1..6195
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     51..73
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     65..82
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     66..82
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     92..111
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        92..110
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             147..350
                     /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             357..872
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     polyA_signal    1003..1124
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1040..1059)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1094..1113
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    1155..1188
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1249..1748
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1284..1307)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1681..1700
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             1809..2828
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             1809..2828
                     /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    3391..3424
                     /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(3473..3493)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3473..3491)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3512..3528)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3512..3528)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3525..3547)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3536..3552
                     /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(3560..3566,3567..3584,3585..3590))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3605..3626
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3743..3760)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3914..4502)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3994..4013)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4673..5464,5465..5533))
                     /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     5296..5315
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5534..5638)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5665..6120
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5752..5771)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5962..5983
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg
       61 acgttgtaaa acgacggcca gtgagcgcgc gtaatacgac tcactatagg gcgaattgga
      121 gctccccggg atccggtgca ggcgccaagg agaagagtgc ttgtcctaaa gatccagcca
      181 aacctccggc caaggcacaa gttgtgggat ggccaccggt gagatcatac cggaagaacg
      241 tgatggtttc ctgccaaaaa tcaagcggtg gcccggaggc ggcggcgttc gtgaaggtat
      301 caatggacgg agcaccgtac ttgaggaaaa tcgatttgag gatgtataaa gctagcatgg
      361 tcttcacact cgaagatttc gttggggact ggcgacagac agccggctac aacctggacc
      421 aagtccttga acagggaggt gtgtccagtt tgtttcagaa tctcggggtg tccgtaactc
      481 cgatccaaag gattgtcctg agcggtgaaa atgggctgaa gatcgacatc catgtcatca
      541 tcccgtatga aggtctgagc ggcgaccaaa tgggccagat cgaaaaaatt tttaaggtgg
      601 tgtaccctgt ggatgatcat cactttaagg tgatcctgca ctatggcaca ctggtaatcg
      661 acggggttac gccgaacatg atcgactatt tcggacggcc gtatgaaggc atcgccgtgt
      721 tcgacggcaa aaagatcact gtaacaggga ccctgtggaa cggcaacaaa attatcgacg
      781 agcgcctgat caaccccgac ggctccctgc tgttccgagt aaccatcaac ggagtgaccg
      841 gctggcggct gtgcgaacgc attctggcgt aagcggccgc tcgagtctag ataactgatc
      901 ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
      961 cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct
     1021 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     1081 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaacgcgt cgatcatatt
     1141 caataaccct taatataact tcgtataatg tatgctatac gaagttatta ggtctgaaga
     1201 ggagtttacg tccagccaag cttaggatct cgacctcgaa attctaccgg gtaggggagg
     1261 cgcttttccc aaggcagtct ggagcatgcg ctttagcagc cccgctgggc acttggcgct
     1321 acacaagtgg cctctggcct cgcacacatt ccacatccac cggtaggcgc caaccgactc
     1381 cgttctttgg tggccccttc gcgccacctt ctactcctcc cctagtcagg aagttccccc
     1441 ccgccccgca gctcgcgtcg tgcaggacgt gacaaatgga agtagcacgt ctcactagtc
     1501 tcgtgcagat ggacagcacc gctgagcaat ggaagcgggt aggcctttgg ggcagcggcc
     1561 aatagcagct ttgctccttc gctttctggg ctcagaggct gggaaggggt gggtccgggg
     1621 gcgggctcag gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc ggaggcccgg
     1681 cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc tcatctccgg
     1741 gcctttcgac ctgcatccat ctagatctcg agcagctgaa gcttaccgct agcatggata
     1801 gatccggaaa gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg
     1861 acagcgtctc cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg
     1921 atgtaggagg gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag
     1981 atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca
     2041 ttggggaatt cagcgagagc ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt
     2101 tgcaagacct gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg gaggccatgg
     2161 atgcgatcgc tgcggccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag
     2221 gaatcggtca atacactaca tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt
     2281 atcactggca aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg
     2341 agctgatgct ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg
     2401 gctccaacaa tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg
     2461 cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg
     2521 cttgtatgga gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc
     2581 cgcggctccg ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg
     2641 acggcaattt cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg
     2701 gagccgggac tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc tggaccgatg
     2761 gctgtgtaga agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa
     2821 aggaataggc tagccgcccg ccccacgacc cgcagcgccc gaccgaaagg agcgcacgac
     2881 cccatgcatc gatgatatca gatccccggg atgcagaaat tgatgatcta ttaaacaata
     2941 aagatgtcca ctaaaatgga agtttttcct gtcatacttt gttaagaagg gtgagaacag
     3001 agtacctaca ttttgaatgg aaggattgga gctacggggg tgggggtggg gtgggattag
     3061 ataaatgcct gctctttact gaaggctctt tactattgct ttatgataat gtttcatagt
     3121 tggatatcat aatttaaaca agcaaaacca aattaagggc cagctcattc ctcccactca
     3181 tgatctatag atctatagat ctctcgtggg atcattgttt ttctcttgat tcccactttg
     3241 tggttctaag tactgtggtt tccaaatgtg tcagtttcat agcctgaaga acgagatcag
     3301 cagcctctgt tccacataca cttcattctc agtattgttt tgccaagttc taattccatc
     3361 agaagctggt cgagatccgg aacccttaat ataacttcgt ataatgtatg ctatacgaag
     3421 ttattaggtc cctcgaagag gttcactagg atccggtacc cagcttttgt tccctttagt
     3481 gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     3541 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     3601 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     3661 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     3721 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     3781 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     3841 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     3901 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     3961 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4021 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4081 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4141 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4201 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4261 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4321 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     4381 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     4441 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4501 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4561 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     4621 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     4681 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     4741 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     4801 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     4861 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     4921 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     4981 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     5041 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     5101 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     5161 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     5221 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     5281 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     5341 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     5401 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     5461 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     5521 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     5581 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     5641 catttccccg aaaagtgcca cctgacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     5701 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     5761 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     5821 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     5881 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     5941 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     6001 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     6061 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     6121 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     6181 attacgccag ctggc
//
