LOCUS       sequence-133565-        6386 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 6386)
  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 6386)
  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: pMK290 (mAID-mClover-Hygro)
FEATURES             Location/Qualifiers
     source          1..6386
                     /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"
     CDS             2534..3553
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             2534..3553
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     protein_bind    4116..4149
                     /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(4198..4218)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4198..4216)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4237..4253)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4237..4253)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4250..4272)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4261..4277
                     /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(4285..4291,4292..4309,4310..4315))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4330..4351
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4468..4485)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4639..5227)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4719..4738)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5398..6189,6190..6258))
                     /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     6021..6040
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6259..6363)
                     /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 ggatagatcc ggaaagcctg aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa
     2581 gttcgacagc gtctccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag
     2641 cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta
     2701 caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc cggaagtgct
     2761 tgacattggg gaattcagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt
     2821 cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc
     2881 catggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc
     2941 gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca
     3001 tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct
     3061 cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga
     3121 tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag
     3181 cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg
     3241 gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg
     3301 atcgccgcgg ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt
     3361 ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg
     3421 atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac
     3481 cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag
     3541 ggcaaaggaa taggctagcc gcccgcccca cgacccgcag cgcccgaccg aaaggagcgc
     3601 acgaccccat gcatcgatga tatcagatcc ccgggatgca gaaattgatg atctattaaa
     3661 caataaagat gtccactaaa atggaagttt ttcctgtcat actttgttaa gaagggtgag
     3721 aacagagtac ctacattttg aatggaagga ttggagctac gggggtgggg gtggggtggg
     3781 attagataaa tgcctgctct ttactgaagg ctctttacta ttgctttatg ataatgtttc
     3841 atagttggat atcataattt aaacaagcaa aaccaaatta agggccagct cattcctccc
     3901 actcatgatc tatagatcta tagatctctc gtgggatcat tgtttttctc ttgattccca
     3961 ctttgtggtt ctaagtactg tggtttccaa atgtgtcagt ttcatagcct gaagaacgag
     4021 atcagcagcc tctgttccac atacacttca ttctcagtat tgttttgcca agttctaatt
     4081 ccatcagaag ctggtcgaga tccggaaccc ttaatataac ttcgtataat gtatgctata
     4141 cgaagttatt aggtccctcg aagaggttca ctaggatccg gtacccagct tttgttccct
     4201 ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     4261 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     4321 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     4381 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4441 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     4501 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     4561 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     4621 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     4681 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4741 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     4801 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     4861 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     4921 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     4981 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5041 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
     5101 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5161 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5221 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     5281 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     5341 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     5401 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     5461 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     5521 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     5581 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     5641 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     5701 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     5761 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     5821 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     5881 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     5941 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     6001 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     6061 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     6121 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     6181 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     6241 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     6301 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     6361 gcgcacattt ccccgaaaag tgccac
//
