LOCUS       sequence-133558-        5566 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 5566)
  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 5566)
  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: pMK279 (mClover-Bsr)
FEATURES             Location/Qualifiers
     source          1..5566
                     /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"
     protein_bind    3296..3329
                     /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(3378..3398)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3378..3396)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3417..3433)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3417..3433)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3430..3452)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3441..3457
                     /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(3465..3471,3472..3489,3490..3495))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3510..3531
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3648..3665)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3819..4407)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3899..3918)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4578..5369,5370..5438))
                     /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     5201..5220
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5439..5543)
                     /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 atgaaaacat ttaacatttc tcaacaagat
     2341 ctagaattag tagaagtagc gacagagaag attacaatgc tttatgagga taataaacat
     2401 catgtgggag cggcaattcg tacgaaaaca ggagaaatca tttcggcagt acatattgaa
     2461 gcgtatatag gacgagtaac tgtttgtgca gaagccattg cgattggtag tgcagtttcg
     2521 aatggacaaa aggattttga cacgattgta gctgttagac acccttattc tgacgaagta
     2581 gatagaagta ttcgagtggt aagtccttgt ggtatgtgta gggagttgat ttcagactat
     2641 gcaccagatt gttttgtgtt aatagaaatg aatggcaagt tagtcaaaac tacgattgaa
     2701 gaactcattc cactcaaata tacccgaaat taagctagcc gcccgcccca cgacccgcag
     2761 cgcccgaccg aaaggagcgc acgaccccat gcatcgatga tatcagatcc ccgggatgca
     2821 gaaattgatg atctattaaa caataaagat gtccactaaa atggaagttt ttcctgtcat
     2881 actttgttaa gaagggtgag aacagagtac ctacattttg aatggaagga ttggagctac
     2941 gggggtgggg gtggggtggg attagataaa tgcctgctct ttactgaagg ctctttacta
     3001 ttgctttatg ataatgtttc atagttggat atcataattt aaacaagcaa aaccaaatta
     3061 agggccagct cattcctccc actcatgatc tatagatcta tagatctctc gtgggatcat
     3121 tgtttttctc ttgattccca ctttgtggtt ctaagtactg tggtttccaa atgtgtcagt
     3181 ttcatagcct gaagaacgag atcagcagcc tctgttccac atacacttca ttctcagtat
     3241 tgttttgcca agttctaatt ccatcagaag ctggtcgaga tccggaaccc ttaatataac
     3301 ttcgtataat gtatgctata cgaagttatt aggtccctcg aagaggttca ctaggatccg
     3361 gtacccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca
     3421 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     3481 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     3541 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     3601 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     3661 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3721 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     3781 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     3841 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3901 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     3961 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4021 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4081 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4141 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4201 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     4261 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4321 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4381 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4441 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     4501 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     4561 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     4621 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     4681 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     4741 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     4801 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     4861 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     4921 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     4981 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     5041 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     5101 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     5161 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     5221 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     5281 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     5341 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     5401 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     5461 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     5521 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccac
//
