LOCUS       sequence-235301-        4168 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 4168)
  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 4168)
  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: pMK407 (BSD-P2A-mAID-Nluc)
FEATURES             Location/Qualifiers
     source          1..4168
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     65..87
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     79..96
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     80..96
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     106..125
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        106..124
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     149..165
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     150..166
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      181..190
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             187..582
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             595..651
                     /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             658..861
                     /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             868..1380
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     primer_bind     complement(1406..1422)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1406..1422)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1459..1479)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1459..1477)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1498..1514)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1498..1514)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1511..1533)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1522..1538
                     /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(1546..1552,1553..1570,1571..1576))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1591..1612
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1729..1746)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1900..2488)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1980..1999)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2659..3450,3451..3519))
                     /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     3282..3301
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3520..3624)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3651..4107
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3738..3757)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3948..3969
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
       61 ttttcccagt cacgacgttg taaaacgacg gccagtgagc gcgcgtaata cgactcacta
      121 tagggcgaat tgggtaccgg gccccccctc gaggtcgacg gtatcgataa gcttgatatc
      181 gccaccatgg ccaagccttt gtctcaagaa gaatccaccc tcattgaaag agcaacggct
      241 acaatcaaca gcatccccat ctctgaagac tacagcgtcg ccagcgcagc tctctctagc
      301 gacggccgca tcttcactgg tgtcaatgta tatcatttta ctgggggacc ttgtgcagaa
      361 ctcgtggtgc tgggcactgc tgctgctgcg gcagctggca acctgacttg tatcgtcgcg
      421 atcggaaatg agaacagggg catcttgagc ccctgcggac ggtgccgaca ggtgcttctc
      481 gatctgcatc ctgggatcaa agccatagtg aaggacagtg atggacagcc gacggcagtt
      541 gggattcgtg aattgctgcc ctctggttat gtgtgggagg gctcaggaag cggagctact
      601 aacttcagcc tgctgaagca ggctggagac gtggaggaga accctggacc tagatctaag
      661 gagaagagtg cttgtcctaa agatccagcc aaacctccgg ccaaggcaca agttgtggga
      721 tggccaccgg tgagatcata ccggaagaac gtgatggttt cctgccaaaa atcaagcggt
      781 ggcccggagg cggcggcgtt cgtgaaggta tcaatggacg gagcaccgta cttgaggaaa
      841 atcgatttga ggatgtataa agctagcatg gtcttcacac tcgaagattt cgttggggac
      901 tggcgacaga cagccggcta caacctggac caagtccttg aacagggagg tgtgtccagt
      961 ttgtttcaga atctcggggt gtccgtaact ccgatccaaa ggattgtcct gagcggtgaa
     1021 aatgggctga agatcgacat ccatgtcatc atcccgtatg aaggtctgag cggcgaccaa
     1081 atgggccaga tcgaaaaaat ttttaaggtg gtgtaccctg tggatgatca tcactttaag
     1141 gtgatcctgc actatggcac actggtaatc gacggggtta cgccgaacat gatcgactat
     1201 ttcggacggc cgtatgaagg catcgccgtg ttcgacggca aaaagatcac tgtaacaggg
     1261 accctgtgga acggcaacaa aattatcgac gagcgcctga tcaaccccga cggctccctg
     1321 ctgttccgag taaccatcaa cggagtgacc ggctggcggc tgtgcgaacg cattctggcg
     1381 ggcgctggtg caggggccgg cgccggatcc actagttcta gagcggccgc caccgcggtg
     1441 gagctccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc
     1501 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     1561 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     1621 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     1681 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     1741 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     1801 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     1861 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     1921 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     1981 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     2041 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     2101 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     2161 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     2221 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     2281 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     2341 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     2401 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     2461 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     2521 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     2581 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     2641 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     2701 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     2761 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     2821 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     2881 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     2941 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     3001 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     3061 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     3121 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     3181 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     3241 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     3301 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     3361 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     3421 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     3481 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     3541 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     3601 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgcgccctg
     3661 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     3721 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     3781 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     3841 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     3901 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     3961 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     4021 gccgatttcg gcctattggt taaaaaaatg agctgattta acaaaaattt aacgcgaatt
     4081 ttaacaaaat attaacgctt acaatttcca ttcgccattc aggctgcgca actgttggga
     4141 agggcgatcg gtgcgggcct cttcgcta
//
