LOCUS       sequence-133554-        5335 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-terminal tagging with Stag-3FLAG using CRIPSR/Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5335)
  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 5335)
  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: pMK283 (Stag-3FLAG-Neo)
FEATURES             Location/Qualifiers
     source          1..5335
                     /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     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             677..721
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             734..799
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    931..1052
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(968..987)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1022..1041
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1059..1514)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        1541..1645
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1647..2004
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1647..1838
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1670..1690)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      1855..1990
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2039..2833
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2039..2833
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2093..2112)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2703..2722
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3021..3040)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     primer_bind     complement(3147..3167)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3147..3165)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3186..3202)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3186..3202)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3199..3221)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3210..3226
                     /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(3234..3240,3241..3258,3259..3264))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3279..3300
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3417..3434)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3588..4176)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3668..3687)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4347..5138,5139..5207))
                     /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     4970..4989
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5208..5312)
                     /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 ggcgccaagg aaaccgccgc tgcaaaattc gaacgccagc acatggacag
      721 ccgatcccgg gctgactaca aagaccatga cggtgattat aaagatcatg acatcgacta
      781 caaggatgac gatgacaagt agtgtacaag taatctagat aactgatcat aatcagccat
      841 accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg
      901 aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac
      961 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     1021 tgtggtttgt ccaaactcat caatgtatct taacgcgtaa attgtaagcg ttaatatttt
     1081 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     1141 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     1201 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     1261 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     1321 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     1381 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     1441 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     1501 gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     1561 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     1621 tcaataatat tgaaaaagga agagtcctga ggcggaaaga accagctgtg gaatgtgtgt
     1681 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     1741 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     1801 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     1861 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     1921 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt
     1981 tttggaggcc taggcttttg caaagatcga tcaagagaca ggatgaggat cgtttcgcat
     2041 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     2101 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     2161 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     2221 agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     2281 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     2341 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     2401 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     2461 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     2521 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg
     2581 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     2641 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     2701 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     2761 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     2821 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     2881 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     2941 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     3001 caccctaggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct
     3061 atgacggcaa taaaaagaca gaataaaacg cacggtgttg ggtcgttgga tccctcgagg
     3121 gggggcccgg tacccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa
     3181 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     3241 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     3301 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     3361 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     3421 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     3481 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     3541 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     3601 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     3661 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     3721 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     3781 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     3841 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     3901 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     3961 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4021 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4081 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4141 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4201 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     4261 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     4321 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     4381 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     4441 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     4501 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     4561 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     4621 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     4681 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     4741 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     4801 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     4861 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     4921 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     4981 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     5041 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     5101 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     5161 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     5221 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     5281 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccac
//
