LOCUS        pMK455 (dTAG-mClover-BSD)        5797 bp    DNA     circular SYN 14-APR-2025
DEFINITION  dTAG-mClover for C-terminal tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5797)
  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 5797)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            //gt12365.cn
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: pMK455 (dTAG-mClover-BSD)
FEATURES             Location/Qualifiers
     source          1..5797
                     /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..1001
                     /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(1014..1016,1017..1019,1020..1733)
                     /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(1059..1080)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1667..1688
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1851..1972
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1888..1907)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1942..1961
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    2003..2036
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2097..2596
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2132..2155)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2529..2548
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      2620..2629
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2626..3024
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     protein_bind    3527..3560
                     /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(3609..3629)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3609..3627)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3648..3664)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3648..3664)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3661..3683)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3672..3688
                     /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(3696..3702,3703..3720,3721..3726))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3741..3762
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3879..3896)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4050..4638)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4130..4149)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4809..5600,5601..5669))
                     /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     5432..5451
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5670..5774)
                     /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 ggagtgcagg tggaaaccat ctccccagga gacgggcgca
      721 ccttccccaa gcgcggccag acctgcgtgg tgcactacac cgggatgctt gaagatggaa
      781 agaaagttga ttcctcccgg gacagaaaca agccctttaa gtttatgcta ggcaagcagg
      841 aggtgatccg aggctgggaa gaaggggttg cccagatgag tgtgggtcag agagccaaac
      901 tgactatatc tccagattat gcctatggtg ccactgggca cccaggcatc atcccaccac
      961 atgccactct cgtcttcgat gtggagcttc taaaactgga aggcggcgct agcatggtga
     1021 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     1081 taaacggcca caagttcagc gtccgcggcg agggcgaggg cgatgccacc aacggcaagc
     1141 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     1201 ccaccttcgg ctacggcgtg gcctgcttca gccgctaccc cgaccacatg aagcagcacg
     1261 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc tctttcaagg
     1321 acgacggtac ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     1381 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     1441 agtacaactt caacagccac aacgtctata tcacggccga caagcagaag aacggcatca
     1501 aggctaactt caagatccgc cacaacgttg aggacggcag cgtgcagctc gccgaccact
     1561 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     1621 gccatcagtc caagctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     1681 agttcgtgac cgccgccggg attacacatg gcatggacga gctgtacaag taatctagat
     1741 aactgatcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc
     1801 cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta
     1861 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     1921 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct taacgcgtcg
     1981 atcatattca ataaccctta atataacttc gtataatgta tgctatacga agttattagg
     2041 tctgaagagg agtttacgtc cagccaagct taggatctcg acctcgaaat tctaccgggt
     2101 aggggaggcg cttttcccaa ggcagtctgg agcatgcgct ttagcagccc cgctgggcac
     2161 ttggcgctac acaagtggcc tctggcctcg cacacattcc acatccaccg gtaggcgcca
     2221 accgactccg ttctttggtg gccccttcgc gccaccttct actcctcccc tagtcaggaa
     2281 gttccccccc gccccgcagc tcgcgtcgtg caggacgtga caaatggaag tagcacgtct
     2341 cactagtctc gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag gcctttgggg
     2401 cagcggccaa tagcagcttt gctccttcgc tttctgggct cagaggctgg gaaggggtgg
     2461 gtccgggggc gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg
     2521 aggcccggca ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc
     2581 atctccgggc ctttcgacct gcatccatct agatctcgag ccaccatggc caagcctttg
     2641 tctcaagaag aatccaccct cattgaaaga gcaacggcta caatcaacag catccccatc
     2701 tctgaagact acagcgtcgc cagcgcagct ctctctagcg acggccgcat cttcactggt
     2761 gtcaatgtat atcattttac tgggggacct tgtgcagaac tcgtggtgct gggcactgct
     2821 gctgctgcgg cagctggcaa cctgacttgt atcgtcgcga tcggaaatga gaacaggggc
     2881 atcttgagcc cctgcggacg gtgccgacag gtgcttctcg atctgcatcc tgggatcaaa
     2941 gccatagtga aggacagtga tggacagccg acggcagttg ggattcgtga attgctgccc
     3001 tctggttatg tgtgggaggg ctaatcgatg atatcagatc cccgggatgc agaaattgat
     3061 gatctattaa acaataaaga tgtccactaa aatggaagtt tttcctgtca tactttgtta
     3121 agaagggtga gaacagagta cctacatttt gaatggaagg attggagcta cgggggtggg
     3181 ggtggggtgg gattagataa atgcctgctc tttactgaag gctctttact attgctttat
     3241 gataatgttt catagttgga tatcataatt taaacaagca aaaccaaatt aagggccagc
     3301 tcattcctcc cactcatgat ctatagatct atagatctct cgtgggatca ttgtttttct
     3361 cttgattccc actttgtggt tctaagtact gtggtttcca aatgtgtcag tttcatagcc
     3421 tgaagaacga gatcagcagc ctctgttcca catacacttc attctcagta ttgttttgcc
     3481 aagttctaat tccatcagaa gctggtcgag atccggaacc cttaatataa cttcgtataa
     3541 tgtatgctat acgaagttat taggtccctc gaagaggttc actaggatcc ggtacccagc
     3601 ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt
     3661 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     3721 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     3781 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     3841 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     3901 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     3961 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4021 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4081 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4141 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4201 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4261 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4321 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4381 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4441 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     4501 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4561 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4621 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4681 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4741 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     4801 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     4861 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     4921 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     4981 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     5041 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     5101 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     5161 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     5221 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     5281 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     5341 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     5401 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     5461 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     5521 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     5581 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     5641 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     5701 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     5761 ataggggttc cgcgcacatt tccccgaaaa gtgccac
//