LOCUS       sequence-331854-        5544 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-terminal tagging with mAID-mClover.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5544)
  AUTHORS   Saito Y, Kanemaki MT
  TITLE     Targeted Protein Depletion Using the Auxin-Inducible Degron 2 (AID2)
            System.
  JOURNAL   Curr Protoc. 2021 Aug;1(8):e219. doi: 10.1002/cpz1.219.
  PUBMED    34370399
REFERENCE   2  (bases 1 to 5544)
  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: "Curr 
            Protoc."; date: "2021-08"; volume: "1(8):e219. doi"; pages: " 
            10.1002/cpz1.219"
COMMENT     Sequence Label: pMK434 (mAID-mClover-P2A-Hygro)
FEATURES             Location/Qualifiers
     source          1..5544
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(85..104)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     295..316
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     579..601
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     593..610
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     594..610
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     620..639
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        620..638
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             675..878
                     /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             join(885..887,888..890,891..1601)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(930..951)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1538..1559
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1608..1664
                     /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             1686..2705
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             1686..2705
                     /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    3268..3301
                     /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(3350..3370)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3350..3368)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3389..3405)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3389..3405)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3402..3424)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3413..3429
                     /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(3437..3443,3444..3461,3462..3467))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3482..3503
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3620..3637)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3791..4379)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3871..3890)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4550..5341,5342..5410))
                     /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     5173..5192
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5411..5515)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(5542..5544,1..453)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
       61 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
      121 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
      181 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
      241 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
      301 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
      361 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
      421 cgaattttaa caaaatatta acgcttacaa tttccattcg ccattcaggc tgcgcaactg
      481 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
      541 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac
      601 gacggccagt gagcgcgcgt aatacgactc actatagggc gaattggagc tccccgggat
      661 ccggtgcagg cgccaaggag aagagtgctt gtcctaaaga tccagccaaa cctccggcca
      721 aggcacaagt tgtgggatgg ccaccggtga gatcataccg gaagaacgtg atggtttcct
      781 gccaaaaatc aagcggtggc ccggaggcgg cggcgttcgt gaaggtatca atggacggag
      841 caccgtactt gaggaaaatc gatttgagga tgtataaagc tagcatggtg agcaagggcg
      901 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
      961 acaagttcag cgtccgcggc gagggcgagg gcgatgccac caacggcaag ctgaccctga
     1021 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccttcg
     1081 gctacggcgt ggcctgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca
     1141 agtccgccat gcccgaaggc tacgtccagg agcgcaccat ctctttcaag gacgacggta
     1201 cctacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
     1261 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     1321 tcaacagcca caacgtctat atcacggccg acaagcagaa gaacggcatc aaggctaact
     1381 tcaagatccg ccacaacgtt gaggacggca gcgtgcagct cgccgaccac taccagcaga
     1441 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agccatcagt
     1501 ccaagctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     1561 ccgccgccgg gattacacat ggcatggacg agctgtacaa gacgcgtgct actaacttca
     1621 gcctgctgaa gcaggctgga gacgtggagg agaaccctgg acctctcgag atggatagat
     1681 ccggaaagcc tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca
     1741 gcgtctccga cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg
     1801 taggagggcg tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc
     1861 gttatgttta tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg
     1921 gggaattcag cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc
     1981 aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg
     2041 cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa
     2101 tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc
     2161 actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc
     2221 tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct
     2281 ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga
     2341 tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt
     2401 gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc
     2461 ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg
     2521 gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag
     2581 ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct
     2641 gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg
     2701 aataggctag ccgcccgccc cacgacccgc agcgcccgac cgaaaggagc gcacgacccc
     2761 atgcatcgat gatatcagat ccccgggatg cagaaattga tgatctatta aacaataaag
     2821 atgtccacta aaatggaagt ttttcctgtc atactttgtt aagaagggtg agaacagagt
     2881 acctacattt tgaatggaag gattggagct acgggggtgg gggtggggtg ggattagata
     2941 aatgcctgct ctttactgaa ggctctttac tattgcttta tgataatgtt tcatagttgg
     3001 atatcataat ttaaacaagc aaaaccaaat taagggccag ctcattcctc ccactcatga
     3061 tctatagatc tatagatctc tcgtgggatc attgtttttc tcttgattcc cactttgtgg
     3121 ttctaagtac tgtggtttcc aaatgtgtca gtttcatagc ctgaagaacg agatcagcag
     3181 cctctgttcc acatacactt cattctcagt attgttttgc caagttctaa ttccatcaga
     3241 agctggtcga gatccggaac ccttaatata acttcgtata atgtatgcta tacgaagtta
     3301 ttaggtccct cgaagaggtt cactaggatc cggtacccag cttttgttcc ctttagtgag
     3361 ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     3421 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     3481 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     3541 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     3601 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     3661 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     3721 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     3781 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     3841 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     3901 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     3961 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4021 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4081 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4141 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4201 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     4261 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4321 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     4381 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     4441 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     4501 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     4561 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     4621 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     4681 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     4741 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     4801 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     4861 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     4921 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     4981 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     5041 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5101 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     5161 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     5221 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     5281 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     5341 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     5401 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5461 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     5521 ttccccgaaa agtgccacct gacg
//
