LOCUS       sequence-235300-        5483 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 5483)
  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 5483)
  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: pMK409 (mAID-Nluc BSD)
FEATURES             Location/Qualifiers
     source          1..5483
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     103..125
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     117..134
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     118..134
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     144..163
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        144..162
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             199..402
                     /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             409..924
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     polyA_signal    1055..1176
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1092..1111)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1146..1165
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    1207..1240
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1301..1800
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1336..1359)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1733..1752
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      1824..1833
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1830..2228
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     protein_bind    2731..2764
                     /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(2813..2833)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2813..2831)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2852..2868)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2852..2868)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2865..2887)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2876..2892
                     /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(2900..2906,2907..2924,2925..2930))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2945..2966
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3083..3100)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3254..3842)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3334..3353)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4013..4804,4805..4873))
                     /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     4636..4655
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4874..4978)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5005..5460
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5092..5111)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5302..5323
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
       61 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
      121 aaacgacggc cagtgagcgc gcgtaatacg actcactata gggcgaattg gagctccccg
      181 ggatccggtg caggcgccaa ggagaagagt gcttgtccta aagatccagc caaacctccg
      241 gccaaggcac aagttgtggg atggccaccg gtgagatcat accggaagaa cgtgatggtt
      301 tcctgccaaa aatcaagcgg tggcccggag gcggcggcgt tcgtgaaggt atcaatggac
      361 ggagcaccgt acttgaggaa aatcgatttg aggatgtata aagctagcat ggtcttcaca
      421 ctcgaagatt tcgttgggga ctggcgacag acagccggct acaacctgga ccaagtcctt
      481 gaacagggag gtgtgtccag tttgtttcag aatctcgggg tgtccgtaac tccgatccaa
      541 aggattgtcc tgagcggtga aaatgggctg aagatcgaca tccatgtcat catcccgtat
      601 gaaggtctga gcggcgacca aatgggccag atcgaaaaaa tttttaaggt ggtgtaccct
      661 gtggatgatc atcactttaa ggtgatcctg cactatggca cactggtaat cgacggggtt
      721 acgccgaaca tgatcgacta tttcggacgg ccgtatgaag gcatcgccgt gttcgacggc
      781 aaaaagatca ctgtaacagg gaccctgtgg aacggcaaca aaattatcga cgagcgcctg
      841 atcaaccccg acggctccct gctgttccga gtaaccatca acggagtgac cggctggcgg
      901 ctgtgcgaac gcattctggc gtaagcggcc gctcgagtct agataactga tcataatcag
      961 ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa
     1021 cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg
     1081 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     1141 tagttgtggt ttgtccaaac tcatcaatgt atcttaacgc gtcgatcata ttcaataacc
     1201 cttaatataa cttcgtataa tgtatgctat acgaagttat taggtctgaa gaggagttta
     1261 cgtccagcca agcttaggat ctcgacctcg aaattctacc gggtagggga ggcgcttttc
     1321 ccaaggcagt ctggagcatg cgctttagca gccccgctgg gcacttggcg ctacacaagt
     1381 ggcctctggc ctcgcacaca ttccacatcc accggtaggc gccaaccgac tccgttcttt
     1441 ggtggcccct tcgcgccacc ttctactcct cccctagtca ggaagttccc ccccgccccg
     1501 cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac gtctcactag tctcgtgcag
     1561 atggacagca ccgctgagca atggaagcgg gtaggccttt ggggcagcgg ccaatagcag
     1621 ctttgctcct tcgctttctg ggctcagagg ctgggaaggg gtgggtccgg gggcgggctc
     1681 aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct ccggaggccc ggcattctgc
     1741 acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt cctcatctcc gggcctttcg
     1801 acctgcatcc atctagatct cgagccacca tggccaagcc tttgtctcaa gaagaatcca
     1861 ccctcattga aagagcaacg gctacaatca acagcatccc catctctgaa gactacagcg
     1921 tcgccagcgc agctctctct agcgacggcc gcatcttcac tggtgtcaat gtatatcatt
     1981 ttactggggg accttgtgca gaactcgtgg tgctgggcac tgctgctgct gcggcagctg
     2041 gcaacctgac ttgtatcgtc gcgatcggaa atgagaacag gggcatcttg agcccctgcg
     2101 gacggtgccg acaggtgctt ctcgatctgc atcctgggat caaagccata gtgaaggaca
     2161 gtgatggaca gccgacggca gttgggattc gtgaattgct gccctctggt tatgtgtggg
     2221 agggctaatc gatgatatca gatccccggg atgcagaaat tgatgatcta ttaaacaata
     2281 aagatgtcca ctaaaatgga agtttttcct gtcatacttt gttaagaagg gtgagaacag
     2341 agtacctaca ttttgaatgg aaggattgga gctacggggg tgggggtggg gtgggattag
     2401 ataaatgcct gctctttact gaaggctctt tactattgct ttatgataat gtttcatagt
     2461 tggatatcat aatttaaaca agcaaaacca aattaagggc cagctcattc ctcccactca
     2521 tgatctatag atctatagat ctctcgtggg atcattgttt ttctcttgat tcccactttg
     2581 tggttctaag tactgtggtt tccaaatgtg tcagtttcat agcctgaaga acgagatcag
     2641 cagcctctgt tccacataca cttcattctc agtattgttt tgccaagttc taattccatc
     2701 agaagctggt cgagatccgg aacccttaat ataacttcgt ataatgtatg ctatacgaag
     2761 ttattaggtc cctcgaagag gttcactagg atccggtacc cagcttttgt tccctttagt
     2821 gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     2881 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     2941 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     3001 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     3061 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     3121 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     3181 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     3241 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     3301 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     3361 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     3421 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     3481 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     3541 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     3601 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     3661 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     3721 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     3781 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     3841 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     3901 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     3961 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     4021 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     4081 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     4141 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     4201 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     4261 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     4321 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     4381 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     4441 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     4501 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     4561 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     4621 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     4681 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     4741 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     4801 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     4861 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     4921 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     4981 catttccccg aaaagtgcca cctgacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     5041 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     5101 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     5161 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     5221 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     5281 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     5341 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     5401 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     5461 ccattcgcca ttcaggctgc gca
//
