LOCUS       sequence-426348-        4489 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dTAG-mClover for N-terminal tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4489)
  AUTHORS   Hatoyama Y, Islam M, Bond AG, Hayashi KI, Ciulli A, Kanemaki MT
  TITLE     Combination of AID2 and BromoTag expands the utility of degron-based
            protein knockdowns.
  JOURNAL   EMBO Rep. 2024 Sep;25(9):4062-4077. doi: 10.1038/s44319-024-00224-4.
            Epub 2024 Aug 23.
  PUBMED    39179892
REFERENCE   2  (bases 1 to 4489)
  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"; doi: 
            "10.1038/s44319-024-00224-4"; journalName: "EMBO Rep"; date: 
            "2024-09"; volume: "25"; issue: "9"; pages: "4062-4077"
COMMENT     Sequence Label: pMK452 (BSD-P2A-dTAG-mClover)
FEATURES             Location/Qualifiers
     source          1..4489
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      4..460
                     /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     586..608
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     600..617
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     601..617
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     627..646
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        627..645
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     670..686
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     671..687
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      702..711
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             708..1103
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             1116..1172
                     /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             1179..1499
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=FKBP-V
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             join(1512..1514,1515..1517,1518..2228)
                     /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(1557..1578)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2165..2186
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2248..2264)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2248..2264)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2301..2321)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2301..2319)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2340..2356)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2340..2356)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2353..2375)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2364..2380
                     /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(2388..2394,2395..2412,2413..2418))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2433..2454
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2571..2588)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2742..3330)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2822..2841)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3501..4292,4293..4361))
                     /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     4124..4143
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4362..4466)
                     /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 atgagctgat ttaacaaaaa
      421 tttaacgcga attttaacaa aatattaacg cttacaattt ccattcgcca ttcaggctgc
      481 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag
      541 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
      601 gtaaaacgac ggccagtgag cgcgcgtaat acgactcact atagggcgaa ttgggtaccg
      661 ggccccccct cgaggtcgac ggtatcgata agcttgatat cgccaccatg gccaagcctt
      721 tgtctcaaga agaatccacc ctcattgaaa gagcaacggc tacaatcaac agcatcccca
      781 tctctgaaga ctacagcgtc gccagcgcag ctctctctag cgacggccgc atcttcactg
      841 gtgtcaatgt atatcatttt actgggggac cttgtgcaga actcgtggtg ctgggcactg
      901 ctgctgctgc ggcagctggc aacctgactt gtatcgtcgc gatcggaaat gagaacaggg
      961 gcatcttgag cccctgcgga cggtgccgac aggtgcttct cgatctgcat cctgggatca
     1021 aagccatagt gaaggacagt gatggacagc cgacggcagt tgggattcgt gaattgctgc
     1081 cctctggtta tgtgtgggag ggctcaggaa gcggagctac taacttcagc ctgctgaagc
     1141 aggctggaga cgtggaggag aaccctggac ctagatctgg agtgcaggtg gaaaccatct
     1201 ccccaggaga cgggcgcacc ttccccaagc gcggccagac ctgcgtggtg cactacaccg
     1261 ggatgcttga agatggaaag aaagttgatt cctcccggga cagaaacaag ccctttaagt
     1321 ttatgctagg caagcaggag gtgatccgag gctgggaaga aggggttgcc cagatgagtg
     1381 tgggtcagag agccaaactg actatatctc cagattatgc ctatggtgcc actgggcacc
     1441 caggcatcat cccaccacat gccactctcg tcttcgatgt ggagcttcta aaactggaag
     1501 gcggcgctag catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     1561 tcgagctgga cggcgacgta aacggccaca agttcagcgt ccgcggcgag ggcgagggcg
     1621 atgccaccaa cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     1681 cctggcccac cctcgtgacc accttcggct acggcgtggc ctgcttcagc cgctaccccg
     1741 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     1801 gcaccatctc tttcaaggac gacggtacct acaagacccg cgccgaggtg aagttcgagg
     1861 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     1921 tcctggggca caagctggag tacaacttca acagccacaa cgtctatatc acggccgaca
     1981 agcagaagaa cggcatcaag gctaacttca agatccgcca caacgttgag gacggcagcg
     2041 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     2101 ccgacaacca ctacctgagc catcagtcca agctgagcaa agaccccaac gagaagcgcg
     2161 atcacatggt cctgctggag ttcgtgaccg ccgccgggat tacacatggc atggacgagc
     2221 tgtacaaggg cgctggtgca ggcgccggat ccactagttc tagagcggcc gccaccgcgg
     2281 tggagctcca gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg
     2341 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     2401 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     2461 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     2521 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     2581 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     2641 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     2701 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     2761 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     2821 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     2881 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     2941 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     3001 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     3061 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     3121 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     3181 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     3241 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     3301 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     3361 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     3421 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     3481 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     3541 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     3601 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     3661 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     3721 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     3781 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     3841 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     3901 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     3961 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     4021 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     4081 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     4141 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     4201 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     4261 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     4321 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     4381 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     4441 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccac
//
