LOCUS       sequence-133551-        4977 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 4977)
  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 4977)
  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: pMK285 (Stag-3FLAG-Bsr)
FEATURES             Location/Qualifiers
     source          1..4977
                     /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"
     protein_bind    2707..2740
                     /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(2789..2809)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2789..2807)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2828..2844)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2828..2844)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2841..2863)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2852..2868
                     /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(2876..2882,2883..2900,2901..2906))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2921..2942
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3059..3076)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3230..3818)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3310..3329)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3989..4780,4781..4849))
                     /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     4612..4631
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4850..4954)
                     /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 catgaaaaca tttaacattt
     1741 ctcaacaaga tctagaatta gtagaagtag cgacagagaa gattacaatg ctttatgagg
     1801 ataataaaca tcatgtggga gcggcaattc gtacgaaaac aggagaaatc atttcggcag
     1861 tacatattga agcgtatata ggacgagtaa ctgtttgtgc agaagccatt gcgattggta
     1921 gtgcagtttc gaatggacaa aaggattttg acacgattgt agctgttaga cacccttatt
     1981 ctgacgaagt agatagaagt attcgagtgg taagtccttg tggtatgtgt agggagttga
     2041 tttcagacta tgcaccagat tgttttgtgt taatagaaat gaatggcaag ttagtcaaaa
     2101 ctacgattga agaactcatt ccactcaaat atacccgaaa ttaagctagc cgcccgcccc
     2161 acgacccgca gcgcccgacc gaaaggagcg cacgacccca tgcatcgatg atatcagatc
     2221 cccgggatgc agaaattgat gatctattaa acaataaaga tgtccactaa aatggaagtt
     2281 tttcctgtca tactttgtta agaagggtga gaacagagta cctacatttt gaatggaagg
     2341 attggagcta cgggggtggg ggtggggtgg gattagataa atgcctgctc tttactgaag
     2401 gctctttact attgctttat gataatgttt catagttgga tatcataatt taaacaagca
     2461 aaaccaaatt aagggccagc tcattcctcc cactcatgat ctatagatct atagatctct
     2521 cgtgggatca ttgtttttct cttgattccc actttgtggt tctaagtact gtggtttcca
     2581 aatgtgtcag tttcatagcc tgaagaacga gatcagcagc ctctgttcca catacacttc
     2641 attctcagta ttgttttgcc aagttctaat tccatcagaa gctggtcgag atccggaacc
     2701 cttaatataa cttcgtataa tgtatgctat acgaagttat taggtccctc gaagaggttc
     2761 actaggatcc ggtacccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt
     2821 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     2881 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     2941 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     3001 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     3061 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     3121 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     3181 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     3241 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3301 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3361 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3421 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3481 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3541 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3601 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     3661 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     3721 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     3781 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     3841 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     3901 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     3961 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     4021 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     4081 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     4141 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     4201 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     4261 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     4321 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     4381 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     4441 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     4501 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     4561 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     4621 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     4681 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     4741 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     4801 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     4861 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     4921 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccac
//
