LOCUS       sequence-133566-        5774 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-terminal tagging with mAID-mClover using CRISPR/Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5774)
  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 5774)
  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: pMK291 (mAID-mClover-Bsr)
FEATURES             Location/Qualifiers
     source          1..5774
                     /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             join(891..893,894..896,897..1610)
                     /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(936..957)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1544..1565
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1728..1849
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1765..1784)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1819..1838
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    1880..1913
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1974..2473
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2009..2032)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2406..2425
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     protein_bind    3504..3537
                     /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(3586..3606)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3586..3604)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3625..3641)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3625..3641)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3638..3660)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3649..3665
                     /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(3673..3679,3680..3697,3698..3703))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3718..3739
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3856..3873)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4027..4615)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4107..4126)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4786..5577,5578..5646))
                     /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     5409..5428
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5647..5751)
                     /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 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
      961 acggccacaa gttcagcgtc cgcggcgagg gcgagggcga tgccaccaac ggcaagctga
     1021 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     1081 ccttcggcta cggcgtggcc tgcttcagcc gctaccccga ccacatgaag cagcacgact
     1141 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatctct ttcaaggacg
     1201 acggtaccta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     1261 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     1321 acaacttcaa cagccacaac gtctatatca cggccgacaa gcagaagaac ggcatcaagg
     1381 ctaacttcaa gatccgccac aacgttgagg acggcagcgt gcagctcgcc gaccactacc
     1441 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagcc
     1501 atcagtccaa gctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     1561 tcgtgaccgc cgccgggatt acacatggca tggacgagct gtacaagtaa tctagataac
     1621 tgatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     1681 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     1741 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     1801 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa cgcgtcgatc
     1861 atattcaata acccttaata taacttcgta taatgtatgc tatacgaagt tattaggtct
     1921 gaagaggagt ttacgtccag ccaagcttag gatctcgacc tcgaaattct accgggtagg
     1981 ggaggcgctt ttcccaaggc agtctggagc atgcgcttta gcagccccgc tgggcacttg
     2041 gcgctacaca agtggcctct ggcctcgcac acattccaca tccaccggta ggcgccaacc
     2101 gactccgttc tttggtggcc ccttcgcgcc accttctact cctcccctag tcaggaagtt
     2161 cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac
     2221 tagtctcgtg cagatggaca gcaccgctga gcaatggaag cgggtaggcc tttggggcag
     2281 cggccaatag cagctttgct ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc
     2341 cgggggcggg ctcaggggcg ggctcagggg cggggcgggc gcccgaaggt cctccggagg
     2401 cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc
     2461 tccgggcctt tcgacctgca tccatctaga tctcgagcag ctgaagctta ccgctagcat
     2521 gaaaacattt aacatttctc aacaagatct agaattagta gaagtagcga cagagaagat
     2581 tacaatgctt tatgaggata ataaacatca tgtgggagcg gcaattcgta cgaaaacagg
     2641 agaaatcatt tcggcagtac atattgaagc gtatatagga cgagtaactg tttgtgcaga
     2701 agccattgcg attggtagtg cagtttcgaa tggacaaaag gattttgaca cgattgtagc
     2761 tgttagacac ccttattctg acgaagtaga tagaagtatt cgagtggtaa gtccttgtgg
     2821 tatgtgtagg gagttgattt cagactatgc accagattgt tttgtgttaa tagaaatgaa
     2881 tggcaagtta gtcaaaacta cgattgaaga actcattcca ctcaaatata cccgaaatta
     2941 agctagccgc ccgccccacg acccgcagcg cccgaccgaa aggagcgcac gaccccatgc
     3001 atcgatgata tcagatcccc gggatgcaga aattgatgat ctattaaaca ataaagatgt
     3061 ccactaaaat ggaagttttt cctgtcatac tttgttaaga agggtgagaa cagagtacct
     3121 acattttgaa tggaaggatt ggagctacgg gggtgggggt ggggtgggat tagataaatg
     3181 cctgctcttt actgaaggct ctttactatt gctttatgat aatgtttcat agttggatat
     3241 cataatttaa acaagcaaaa ccaaattaag ggccagctca ttcctcccac tcatgatcta
     3301 tagatctata gatctctcgt gggatcattg tttttctctt gattcccact ttgtggttct
     3361 aagtactgtg gtttccaaat gtgtcagttt catagcctga agaacgagat cagcagcctc
     3421 tgttccacat acacttcatt ctcagtattg ttttgccaag ttctaattcc atcagaagct
     3481 ggtcgagatc cggaaccctt aatataactt cgtataatgt atgctatacg aagttattag
     3541 gtccctcgaa gaggttcact aggatccggt acccagcttt tgttcccttt agtgagggtt
     3601 aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     3661 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     3721 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     3781 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     3841 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     3901 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     3961 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     4021 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     4081 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     4141 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     4201 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     4261 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     4321 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     4381 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     4441 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
     4501 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     4561 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     4621 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     4681 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     4741 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     4801 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     4861 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     4921 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     4981 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     5041 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     5101 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     5161 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     5221 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     5281 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     5341 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     5401 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     5461 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     5521 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     5581 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     5641 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     5701 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     5761 ccgaaaagtg ccac
//
