LOCUS       pMK456 (dTAG-mCherry-BSD)       5788 bp    DNA     circular SYN 14-APR-2025
DEFINITION  dTAG-mCherry for C-terminal tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5788)
  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 5788)
  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: pMK456 (dTAG-mCherry-BSD)
FEATURES             Location/Qualifiers
     source          1..5788
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      3..458
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(90..109)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     300..321
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     584..606
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     598..615
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     599..615
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     625..644
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        625..643
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             680..1000
                     /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             1013..1723
                     /codon_start=1
                     /product="derivative of the monomeric red fluorescent 
                     protein mCherry"
                     /label=mCherry2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFNWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQCRTMGWEASTERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVDIKLDILSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1158..1176)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    1841..1962
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1878..1897)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1932..1951
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    1993..2026
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2087..2586
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2122..2145)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2519..2538
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      2610..2619
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2616..3014
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     protein_bind    3517..3550
                     /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(3599..3619)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3599..3617)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3638..3654)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3638..3654)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3651..3673)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3662..3678
                     /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(3686..3692,3693..3710,3711..3716))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3731..3752
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3869..3886)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4040..4628)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4120..4139)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4799..5590,5591..5659))
                     /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     5422..5441
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5660..5764)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tgacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
       61 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
      121 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
      181 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
      241 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
      301 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
      361 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
      421 taacgcgaat tttaacaaaa tattaacgct tacaatttcc attcgccatt caggctgcgc
      481 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg
      541 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt
      601 aaaacgacgg ccagtgagcg cgcgtaatac gactcactat agggcgaatt ggagctcccc
      661 gggatccggt gcaggcgccg gagtgcaggt ggaaaccatc tccccaggag acgggcgcac
      721 cttccccaag cgcggccaga cctgcgtggt gcactacacc gggatgcttg aagatggaaa
      781 gaaagttgat tcctcccggg acagaaacaa gccctttaag tttatgctag gcaagcagga
      841 ggtgatccga ggctgggaag aaggggttgc ccagatgagt gtgggtcaga gagccaaact
      901 gactatatct ccagattatg cctatggtgc cactgggcac ccaggcatca tcccaccaca
      961 tgccactctc gtcttcgatg tggagcttct aaaactggaa ggcggcgcta gcatggtgag
     1021 caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat
     1081 ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     1141 cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg
     1201 ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga
     1261 catccccgac tacttgaagc tgtccttccc cgagggcttc aattgggagc gcgtgatgaa
     1321 cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt
     1381 catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagtg
     1441 tcgtaccatg ggctgggagg cctccactga gcggatgtac cccgaggacg gcgccctgaa
     1501 gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa
     1561 gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcgacatcaa
     1621 gttggacatc ctttcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga
     1681 gggccgccac tccaccggcg gcatggacga gctgtacaag taatctagat aactgatcat
     1741 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     1801 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
     1861 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     1921 gcattctagt tgtggtttgt ccaaactcat caatgtatct taacgcgtcg atcatattca
     1981 ataaccctta atataacttc gtataatgta tgctatacga agttattagg tctgaagagg
     2041 agtttacgtc cagccaagct taggatctcg acctcgaaat tctaccgggt aggggaggcg
     2101 cttttcccaa ggcagtctgg agcatgcgct ttagcagccc cgctgggcac ttggcgctac
     2161 acaagtggcc tctggcctcg cacacattcc acatccaccg gtaggcgcca accgactccg
     2221 ttctttggtg gccccttcgc gccaccttct actcctcccc tagtcaggaa gttccccccc
     2281 gccccgcagc tcgcgtcgtg caggacgtga caaatggaag tagcacgtct cactagtctc
     2341 gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag gcctttgggg cagcggccaa
     2401 tagcagcttt gctccttcgc tttctgggct cagaggctgg gaaggggtgg gtccgggggc
     2461 gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg aggcccggca
     2521 ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc atctccgggc
     2581 ctttcgacct gcatccatct agatctcgag ccaccatggc caagcctttg tctcaagaag
     2641 aatccaccct cattgaaaga gcaacggcta caatcaacag catccccatc tctgaagact
     2701 acagcgtcgc cagcgcagct ctctctagcg acggccgcat cttcactggt gtcaatgtat
     2761 atcattttac tgggggacct tgtgcagaac tcgtggtgct gggcactgct gctgctgcgg
     2821 cagctggcaa cctgacttgt atcgtcgcga tcggaaatga gaacaggggc atcttgagcc
     2881 cctgcggacg gtgccgacag gtgcttctcg atctgcatcc tgggatcaaa gccatagtga
     2941 aggacagtga tggacagccg acggcagttg ggattcgtga attgctgccc tctggttatg
     3001 tgtgggaggg ctaatcgatg atatcagatc cccgggatgc agaaattgat gatctattaa
     3061 acaataaaga tgtccactaa aatggaagtt tttcctgtca tactttgtta agaagggtga
     3121 gaacagagta cctacatttt gaatggaagg attggagcta cgggggtggg ggtggggtgg
     3181 gattagataa atgcctgctc tttactgaag gctctttact attgctttat gataatgttt
     3241 catagttgga tatcataatt taaacaagca aaaccaaatt aagggccagc tcattcctcc
     3301 cactcatgat ctatagatct atagatctct cgtgggatca ttgtttttct cttgattccc
     3361 actttgtggt tctaagtact gtggtttcca aatgtgtcag tttcatagcc tgaagaacga
     3421 gatcagcagc ctctgttcca catacacttc attctcagta ttgttttgcc aagttctaat
     3481 tccatcagaa gctggtcgag atccggaacc cttaatataa cttcgtataa tgtatgctat
     3541 acgaagttat taggtccctc gaagaggttc actaggatcc ggtacccagc ttttgttccc
     3601 tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     3661 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     3721 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     3781 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     3841 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     3901 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     3961 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4021 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4081 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4141 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4201 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4261 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4321 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4381 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4441 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     4501 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4561 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4621 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4681 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     4741 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     4801 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     4861 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     4921 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     4981 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5041 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5101 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5161 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5221 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5281 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5341 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5401 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     5461 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     5521 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     5581 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     5641 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     5701 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     5761 cgcgcacatt tccccgaaaa gtgccacc
//