LOCUS       sequence-196921-        5663 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-terminal tagging with mAID using CRISPR/Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5663)
  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 5663)
  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: pMK287 (mAID-Hygro)
FEATURES             Location/Qualifiers
     source          1..5663
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    454..487
                     /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(536..556)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(536..554)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(575..591)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(575..591)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(588..610)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    599..615
                     /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(623..629,630..647,648..653))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    668..689
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(806..823)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(977..1565)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1057..1076)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1736..2527,2528..2596))
                     /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     2359..2378
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2597..2701)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2728..3183
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2815..2834)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3025..3046
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3309..3331
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3323..3340
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3324..3340
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3350..3369
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3350..3368
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3405..3608
                     /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"
     polyA_signal    3729..3850
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3766..3785)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3820..3839
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    3881..3914
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3975..4474
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4010..4033)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4407..4426
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             4535..5554
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             4535..5554
                     /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"
ORIGIN
        1 ataaagatgt ccactaaaat ggaagttttt cctgtcatac tttgttaaga agggtgagaa
       61 cagagtacct acattttgaa tggaaggatt ggagctacgg gggtgggggt ggggtgggat
      121 tagataaatg cctgctcttt actgaaggct ctttactatt gctttatgat aatgtttcat
      181 agttggatat cataatttaa acaagcaaaa ccaaattaag ggccagctca ttcctcccac
      241 tcatgatcta tagatctata gatctctcgt gggatcattg tttttctctt gattcccact
      301 ttgtggttct aagtactgtg gtttccaaat gtgtcagttt catagcctga agaacgagat
      361 cagcagcctc tgttccacat acacttcatt ctcagtattg ttttgccaag ttctaattcc
      421 atcagaagct ggtcgagatc cggaaccctt aatataactt cgtataatgt atgctatacg
      481 aagttattag gtccctcgaa gaggttcact aggatccggt acccagcttt tgttcccttt
      541 agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
      601 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
      661 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
      721 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
      781 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
      841 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
      901 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
      961 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     1021 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     1081 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     1141 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     1201 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     1261 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     1321 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     1381 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     1441 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     1501 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     1561 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     1621 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     1681 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     1741 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     1801 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     1861 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     1921 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     1981 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     2041 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     2101 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     2161 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     2221 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     2281 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     2341 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     2401 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     2461 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     2521 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     2581 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     2641 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     2701 gcacatttcc ccgaaaagtg ccacctgacg cgccctgtag cggcgcatta agcgcggcgg
     2761 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     2821 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     2881 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     2941 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     3001 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     3061 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     3121 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     3181 tttccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
     3241 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
     3301 agggttttcc cagtcacgac gttgtaaaac gacggccagt gagcgcgcgt aatacgactc
     3361 actatagggc gaattggagc tccccgggat ccggtgcagg cgccaaggag aagagtgctt
     3421 gtcctaaaga tccagccaaa cctccggcca aggcacaagt tgtgggatgg ccaccggtga
     3481 gatcataccg gaagaacgtg atggtttcct gccaaaaatc aagcggtggc ccggaggcgg
     3541 cggcgttcgt gaaggtatca atggacggag caccgtactt gaggaaaatc gatttgagga
     3601 tgtataaata atctagataa ctgatcataa tcagccatac cacatttgta gaggttttac
     3661 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
     3721 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     3781 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     3841 atgtatctta acgcgtcgat catattcaat aacccttaat ataacttcgt ataatgtatg
     3901 ctatacgaag ttattaggtc tgaagaggag tttacgtcca gccaagctta ggatctcgac
     3961 ctcgaaattc taccgggtag gggaggcgct tttcccaagg cagtctggag catgcgcttt
     4021 agcagccccg ctgggcactt ggcgctacac aagtggcctc tggcctcgca cacattccac
     4081 atccaccggt aggcgccaac cgactccgtt ctttggtggc cccttcgcgc caccttctac
     4141 tcctccccta gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca
     4201 aatggaagta gcacgtctca ctagtctcgt gcagatggac agcaccgctg agcaatggaa
     4261 gcgggtaggc ctttggggca gcggccaata gcagctttgc tccttcgctt tctgggctca
     4321 gaggctggga aggggtgggt ccgggggcgg gctcaggggc gggctcaggg gcggggcggg
     4381 cgcccgaagg tcctccggag gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc
     4441 gctgttctcc tcttcctcat ctccgggcct ttcgacctgc atccatctag atctcgagca
     4501 gctgaagctt accgctagca tggatagatc cggaaagcct gaactcaccg cgacgtctgt
     4561 cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg
     4621 cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa
     4681 tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc
     4741 gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat
     4801 ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt
     4861 tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag
     4921 cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat
     4981 atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag
     5041 tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt
     5101 ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat
     5161 aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa
     5221 catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg
     5281 gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct
     5341 tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg
     5401 tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg
     5461 cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg
     5521 acgccccagc actcgtccga gggcaaagga ataggctagc cgcccgcccc acgacccgca
     5581 gcgcccgacc gaaaggagcg cacgacccca tgcatcgatg atatcagatc cccgggatgc
     5641 agaaattgat gatctattaa aca
//
