LOCUS       sequence-133553-        5589 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 5589)
  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 5589)
  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: pMK284 (Stag-3FLAG-Hygro)
FEATURES             Location/Qualifiers
     source          1..5589
                     /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             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"
     protein_bind    1083..1116
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1177..1676
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1212..1235)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1609..1628
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             1737..2756
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             1737..2756
                     /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    3319..3352
                     /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(3401..3421)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3401..3419)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3440..3456)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3440..3456)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3453..3475)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3464..3480
                     /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(3488..3494,3495..3512,3513..3518))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3533..3554
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3671..3688)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3842..4430)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3922..3941)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4601..5392,5393..5461))
                     /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     5224..5243
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5462..5566)
                     /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 taacgcgtcg atcatattca ataaccctta
     1081 atataacttc gtataatgta tgctatacga agttattagg tctgaagagg agtttacgtc
     1141 cagccaagct taggatctcg acctcgaaat tctaccgggt aggggaggcg cttttcccaa
     1201 ggcagtctgg agcatgcgct ttagcagccc cgctgggcac ttggcgctac acaagtggcc
     1261 tctggcctcg cacacattcc acatccaccg gtaggcgcca accgactccg ttctttggtg
     1321 gccccttcgc gccaccttct actcctcccc tagtcaggaa gttccccccc gccccgcagc
     1381 tcgcgtcgtg caggacgtga caaatggaag tagcacgtct cactagtctc gtgcagatgg
     1441 acagcaccgc tgagcaatgg aagcgggtag gcctttgggg cagcggccaa tagcagcttt
     1501 gctccttcgc tttctgggct cagaggctgg gaaggggtgg gtccgggggc gggctcaggg
     1561 gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg aggcccggca ttctgcacgc
     1621 ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc atctccgggc ctttcgacct
     1681 gcatccatct agatctcgag cagctgaagc ttaccgctag catggataga tccggaaagc
     1741 ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg
     1801 acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc
     1861 gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt
     1921 atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattca
     1981 gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc
     2041 ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg
     2101 cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat
     2161 acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa
     2221 ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt
     2281 gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg
     2341 tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg
     2401 attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc
     2461 agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg
     2521 cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg
     2581 atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg
     2641 tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag
     2701 tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaataggcta
     2761 gccgcccgcc ccacgacccg cagcgcccga ccgaaaggag cgcacgaccc catgcatcga
     2821 tgatatcaga tccccgggat gcagaaattg atgatctatt aaacaataaa gatgtccact
     2881 aaaatggaag tttttcctgt catactttgt taagaagggt gagaacagag tacctacatt
     2941 ttgaatggaa ggattggagc tacgggggtg ggggtggggt gggattagat aaatgcctgc
     3001 tctttactga aggctcttta ctattgcttt atgataatgt ttcatagttg gatatcataa
     3061 tttaaacaag caaaaccaaa ttaagggcca gctcattcct cccactcatg atctatagat
     3121 ctatagatct ctcgtgggat cattgttttt ctcttgattc ccactttgtg gttctaagta
     3181 ctgtggtttc caaatgtgtc agtttcatag cctgaagaac gagatcagca gcctctgttc
     3241 cacatacact tcattctcag tattgttttg ccaagttcta attccatcag aagctggtcg
     3301 agatccggaa cccttaatat aacttcgtat aatgtatgct atacgaagtt attaggtccc
     3361 tcgaagaggt tcactaggat ccggtaccca gcttttgttc cctttagtga gggttaattg
     3421 cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     3481 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     3541 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3601 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     3661 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3721 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3781 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3841 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3901 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3961 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4021 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4081 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4141 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4201 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4261 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     4321 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4381 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4441 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4501 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     4561 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4621 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4681 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4741 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     4801 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     4861 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     4921 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4981 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5041 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     5101 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     5161 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     5221 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     5281 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     5341 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     5401 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     5461 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     5521 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     5581 aagtgccac
//
