LOCUS       sequence-230649-        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"
     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             681..884
                     /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             891..1406
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     polyA_signal    1537..1658
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1574..1593)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1628..1647
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    1689..1722
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1783..2282
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1818..1841)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2215..2234
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2343..3362
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             2343..3362
                     /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    3925..3958
                     /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(4007..4027)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4007..4025)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4046..4062)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4046..4062)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4059..4081)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4070..4086
                     /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(4094..4100,4101..4118,4119..4124))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4139..4160
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4277..4294)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4448..5036)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4528..4547)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5207..5998,5999..6067))
                     /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     5830..5849
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6068..6172)
                     /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 tgcaggcgcc aaggagaaga gtgcttgtcc taaagatcca gccaaacctc
      721 cggccaaggc acaagttgtg ggatggccac cggtgagatc ataccggaag aacgtgatgg
      781 tttcctgcca aaaatcaagc ggtggcccgg aggcggcggc gttcgtgaag gtatcaatgg
      841 acggagcacc gtacttgagg aaaatcgatt tgaggatgta taaagctagc atggtcttca
      901 cactcgaaga tttcgttggg gactggcgac agacagccgg ctacaacctg gaccaagtcc
      961 ttgaacaggg aggtgtgtcc agtttgtttc agaatctcgg ggtgtccgta actccgatcc
     1021 aaaggattgt cctgagcggt gaaaatgggc tgaagatcga catccatgtc atcatcccgt
     1081 atgaaggtct gagcggcgac caaatgggcc agatcgaaaa aatttttaag gtggtgtacc
     1141 ctgtggatga tcatcacttt aaggtgatcc tgcactatgg cacactggta atcgacgggg
     1201 ttacgccgaa catgatcgac tatttcggac ggccgtatga aggcatcgcc gtgttcgacg
     1261 gcaaaaagat cactgtaaca gggaccctgt ggaacggcaa caaaattatc gacgagcgcc
     1321 tgatcaaccc cgacggctcc ctgctgttcc gagtaaccat caacggagtg accggctggc
     1381 ggctgtgcga acgcattctg gcgtaagcgg ccgctcgagt ctagataact gatcataatc
     1441 agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg
     1501 aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat
     1561 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     1621 tctagttgtg gtttgtccaa actcatcaat gtatcttaac gcgtcgatca tattcaataa
     1681 cccttaatat aacttcgtat aatgtatgct atacgaagtt attaggtctg aagaggagtt
     1741 tacgtccagc caagcttagg atctcgacct cgaaattcta ccgggtaggg gaggcgcttt
     1801 tcccaaggca gtctggagca tgcgctttag cagccccgct gggcacttgg cgctacacaa
     1861 gtggcctctg gcctcgcaca cattccacat ccaccggtag gcgccaaccg actccgttct
     1921 ttggtggccc cttcgcgcca ccttctactc ctcccctagt caggaagttc ccccccgccc
     1981 cgcagctcgc gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact agtctcgtgc
     2041 agatggacag caccgctgag caatggaagc gggtaggcct ttggggcagc ggccaatagc
     2101 agctttgctc cttcgctttc tgggctcaga ggctgggaag gggtgggtcc gggggcgggc
     2161 tcaggggcgg gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc ccggcattct
     2221 gcacgcttca aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct ccgggccttt
     2281 cgacctgcat ccatctagat ctcgagcagc tgaagcttac cgctagcatg gatagatccg
     2341 gaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg
     2401 tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag
     2461 gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt
     2521 atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg
     2581 aattcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag
     2641 acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc atggatgcga
     2701 tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg
     2761 gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact
     2821 ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga
     2881 tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca
     2941 acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt
     3001 tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta
     3061 tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc
     3121 tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca
     3181 atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg
     3241 ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg
     3301 tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat
     3361 aggctagccg cccgccccac gacccgcagc gcccgaccga aaggagcgca cgaccccatg
     3421 catcgatgat atcagatccc cgggatgcag aaattgatga tctattaaac aataaagatg
     3481 tccactaaaa tggaagtttt tcctgtcata ctttgttaag aagggtgaga acagagtacc
     3541 tacattttga atggaaggat tggagctacg ggggtggggg tggggtggga ttagataaat
     3601 gcctgctctt tactgaaggc tctttactat tgctttatga taatgtttca tagttggata
     3661 tcataattta aacaagcaaa accaaattaa gggccagctc attcctccca ctcatgatct
     3721 atagatctat agatctctcg tgggatcatt gtttttctct tgattcccac tttgtggttc
     3781 taagtactgt ggtttccaaa tgtgtcagtt tcatagcctg aagaacgaga tcagcagcct
     3841 ctgttccaca tacacttcat tctcagtatt gttttgccaa gttctaattc catcagaagc
     3901 tggtcgagat ccggaaccct taatataact tcgtataatg tatgctatac gaagttatta
     3961 ggtccctcga agaggttcac taggatccgg tacccagctt ttgttccctt tagtgagggt
     4021 taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     4081 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     4141 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     4201 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     4261 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     4321 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     4381 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     4441 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     4501 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     4561 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     4621 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     4681 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     4741 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4801 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4861 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     4921 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4981 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     5041 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     5101 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     5161 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     5221 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     5281 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     5341 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     5401 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     5461 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     5521 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     5581 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     5641 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     5701 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     5761 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     5821 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     5881 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     5941 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     6001 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     6061 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     6121 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     6181 cccgaaaagt gccac
//
