LOCUS       sequence-133559-        6178 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-terminal tagging with mClover using CRIPSR/Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6178)
  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 6178)
  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: pMK278 (mClover-Hygro)
FEATURES             Location/Qualifiers
     source          1..6178
                     /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             join(683..685,686..688,689..1402)
                     /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(728..749)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1336..1357
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1520..1641
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1557..1576)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1611..1630
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    1672..1705
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1766..2265
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1801..1824)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2198..2217
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2326..3345
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             2326..3345
                     /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    3908..3941
                     /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(3990..4010)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3990..4008)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4029..4045)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4029..4045)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4042..4064)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4053..4069
                     /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(4077..4083,4084..4101,4102..4107))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4122..4143
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4260..4277)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4431..5019)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4511..4530)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5190..5981,5982..6050))
                     /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     5813..5832
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6051..6155)
                     /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 tggagctcgg
      661 atccggtgca ggcgccgcta gcatggtgag caagggcgag gagctgttca ccggggtggt
      721 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tccgcggcga
      781 gggcgagggc gatgccacca acggcaagct gaccctgaag ttcatctgca ccaccggcaa
      841 gctgcccgtg ccctggccca ccctcgtgac caccttcggc tacggcgtgg cctgcttcag
      901 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
      961 cgtccaggag cgcaccatct ctttcaagga cgacggtacc tacaagaccc gcgccgaggt
     1021 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     1081 ggacggcaac atcctggggc acaagctgga gtacaacttc aacagccaca acgtctatat
     1141 cacggccgac aagcagaaga acggcatcaa ggctaacttc aagatccgcc acaacgttga
     1201 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     1261 cgtgctgctg cccgacaacc actacctgag ccatcagtcc aagctgagca aagaccccaa
     1321 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga ttacacatgg
     1381 catggacgag ctgtacaagt aatctagata actgatcata atcagccata ccacatttgt
     1441 agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat
     1501 gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa
     1561 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     1621 caaactcatc aatgtatctt aacgcgtcga tcatattcaa taacccttaa tataacttcg
     1681 tataatgtat gctatacgaa gttattaggt ctgaagagga gtttacgtcc agccaagctt
     1741 aggatctcga cctcgaaatt ctaccgggta ggggaggcgc ttttcccaag gcagtctgga
     1801 gcatgcgctt tagcagcccc gctgggcact tggcgctaca caagtggcct ctggcctcgc
     1861 acacattcca catccaccgg taggcgccaa ccgactccgt tctttggtgg ccccttcgcg
     1921 ccaccttcta ctcctcccct agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc
     1981 aggacgtgac aaatggaagt agcacgtctc actagtctcg tgcagatgga cagcaccgct
     2041 gagcaatgga agcgggtagg cctttggggc agcggccaat agcagctttg ctccttcgct
     2101 ttctgggctc agaggctggg aaggggtggg tccgggggcg ggctcagggg cgggctcagg
     2161 ggcggggcgg gcgcccgaag gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc
     2221 acgtctgccg cgctgttctc ctcttcctca tctccgggcc tttcgacctg catccatcta
     2281 gatctcgagc agctgaagct taccgctagc atggatagat ccggaaagcc tgaactcacc
     2341 gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag
     2401 ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc
     2461 ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt
     2521 gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg
     2581 acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa
     2641 ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt
     2701 agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg
     2761 cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac
     2821 gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac
     2881 tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac
     2941 aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac
     3001 gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc
     3061 tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc
     3121 cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct
     3181 tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca
     3241 caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat
     3301 agtggaaacc gacgccccag cactcgtccg agggcaaagg aataggctag ccgcccgccc
     3361 cacgacccgc agcgcccgac cgaaaggagc gcacgacccc atgcatcgat gatatcagat
     3421 ccccgggatg cagaaattga tgatctatta aacaataaag atgtccacta aaatggaagt
     3481 ttttcctgtc atactttgtt aagaagggtg agaacagagt acctacattt tgaatggaag
     3541 gattggagct acgggggtgg gggtggggtg ggattagata aatgcctgct ctttactgaa
     3601 ggctctttac tattgcttta tgataatgtt tcatagttgg atatcataat ttaaacaagc
     3661 aaaaccaaat taagggccag ctcattcctc ccactcatga tctatagatc tatagatctc
     3721 tcgtgggatc attgtttttc tcttgattcc cactttgtgg ttctaagtac tgtggtttcc
     3781 aaatgtgtca gtttcatagc ctgaagaacg agatcagcag cctctgttcc acatacactt
     3841 cattctcagt attgttttgc caagttctaa ttccatcaga agctggtcga gatccggaac
     3901 ccttaatata acttcgtata atgtatgcta tacgaagtta ttaggtccct cgaagaggtt
     3961 cactaggatc cggtacccag cttttgttcc ctttagtgag ggttaattgc gcgcttggcg
     4021 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     4081 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     4141 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     4201 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     4261 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     4321 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     4381 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     4441 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     4501 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     4561 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4621 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4681 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4741 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4801 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4861 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4921 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4981 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     5041 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     5101 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     5161 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     5221 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     5281 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     5341 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     5401 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     5461 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     5521 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     5581 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     5641 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     5701 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     5761 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     5821 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     5881 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     5941 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     6001 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     6061 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     6121 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccac
//
