LOCUS       sequence-133569-        5765 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-terminal tagging with mAID-mCherry2 using CRISPR/Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5765)
  AUTHORS   Natsume T, Kiyomitsu T, Saga Y, Kanemaki MT
  TITLE     Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron 
            Tagging with Short Homology Donors.
  JOURNAL   Cell Rep. 2016 Apr 5;15(1):210-8. doi: 10.1016/j.celrep.2016.03.001.
            Epub 2016 Mar 24.
  PUBMED    27052166
REFERENCE   2  (bases 1 to 5765)
  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: 
            "10.1016/j.celrep.2016.03.001"; journalName: "Cell Rep"; date: 
            "2016-04-5- 5"; volume: "15"; issue: "1"; pages: "210-8"
COMMENT     Sequence Label: pMK294 (mAID-mCherry2-Bsr)
FEATURES             Location/Qualifiers
     source          1..5765
                     /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..1601
                     /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(1036..1054)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    1719..1840
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1756..1775)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1810..1829
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    1871..1904
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1965..2464
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2000..2023)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2397..2416
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     protein_bind    3495..3528
                     /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(3577..3597)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3577..3595)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3616..3632)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3616..3632)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3629..3651)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3640..3656
                     /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(3664..3670,3671..3688,3689..3694))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3709..3730
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3847..3864)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4018..4606)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4098..4117)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4777..5568,5569..5637))
                     /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     5400..5419
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5638..5742)
                     /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 atggtgagca
      901 agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg
      961 agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
     1021 agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg
     1081 acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca
     1141 tccccgacta cttgaagctg tccttccccg agggcttcaa ttgggagcgc gtgatgaact
     1201 tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca
     1261 tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagtgtc
     1321 gtaccatggg ctgggaggcc tccactgagc ggatgtaccc cgaggacggc gccctgaagg
     1381 gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct gaggtcaaga
     1441 ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc gacatcaagt
     1501 tggacatcct ttcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg
     1561 gccgccactc caccggcggc atggacgagc tgtacaagta atctagataa ctgatcataa
     1621 tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc
     1681 tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata
     1741 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     1801 attctagttg tggtttgtcc aaactcatca atgtatctta acgcgtcgat catattcaat
     1861 aacccttaat ataacttcgt ataatgtatg ctatacgaag ttattaggtc tgaagaggag
     1921 tttacgtcca gccaagctta ggatctcgac ctcgaaattc taccgggtag gggaggcgct
     1981 tttcccaagg cagtctggag catgcgcttt agcagccccg ctgggcactt ggcgctacac
     2041 aagtggcctc tggcctcgca cacattccac atccaccggt aggcgccaac cgactccgtt
     2101 ctttggtggc cccttcgcgc caccttctac tcctccccta gtcaggaagt tcccccccgc
     2161 cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta gcacgtctca ctagtctcgt
     2221 gcagatggac agcaccgctg agcaatggaa gcgggtaggc ctttggggca gcggccaata
     2281 gcagctttgc tccttcgctt tctgggctca gaggctggga aggggtgggt ccgggggcgg
     2341 gctcaggggc gggctcaggg gcggggcggg cgcccgaagg tcctccggag gcccggcatt
     2401 ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc tcttcctcat ctccgggcct
     2461 ttcgacctgc atccatctag atctcgagca gctgaagctt accgctagca tgaaaacatt
     2521 taacatttct caacaagatc tagaattagt agaagtagcg acagagaaga ttacaatgct
     2581 ttatgaggat aataaacatc atgtgggagc ggcaattcgt acgaaaacag gagaaatcat
     2641 ttcggcagta catattgaag cgtatatagg acgagtaact gtttgtgcag aagccattgc
     2701 gattggtagt gcagtttcga atggacaaaa ggattttgac acgattgtag ctgttagaca
     2761 cccttattct gacgaagtag atagaagtat tcgagtggta agtccttgtg gtatgtgtag
     2821 ggagttgatt tcagactatg caccagattg ttttgtgtta atagaaatga atggcaagtt
     2881 agtcaaaact acgattgaag aactcattcc actcaaatat acccgaaatt aagctagccg
     2941 cccgccccac gacccgcagc gcccgaccga aaggagcgca cgaccccatg catcgatgat
     3001 atcagatccc cgggatgcag aaattgatga tctattaaac aataaagatg tccactaaaa
     3061 tggaagtttt tcctgtcata ctttgttaag aagggtgaga acagagtacc tacattttga
     3121 atggaaggat tggagctacg ggggtggggg tggggtggga ttagataaat gcctgctctt
     3181 tactgaaggc tctttactat tgctttatga taatgtttca tagttggata tcataattta
     3241 aacaagcaaa accaaattaa gggccagctc attcctccca ctcatgatct atagatctat
     3301 agatctctcg tgggatcatt gtttttctct tgattcccac tttgtggttc taagtactgt
     3361 ggtttccaaa tgtgtcagtt tcatagcctg aagaacgaga tcagcagcct ctgttccaca
     3421 tacacttcat tctcagtatt gttttgccaa gttctaattc catcagaagc tggtcgagat
     3481 ccggaaccct taatataact tcgtataatg tatgctatac gaagttatta ggtccctcga
     3541 agaggttcac taggatccgg tacccagctt ttgttccctt tagtgagggt taattgcgcg
     3601 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     3661 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     3721 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     3781 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     3841 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     3901 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     3961 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     4021 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     4081 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4141 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     4201 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     4261 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     4321 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     4381 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     4441 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     4501 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     4561 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     4621 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     4681 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     4741 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     4801 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     4861 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     4921 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     4981 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     5041 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     5101 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     5161 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     5221 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     5281 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     5341 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     5401 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     5461 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     5521 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     5581 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     5641 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     5701 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     5761 gccac
//
