LOCUS       sequence-230639-        4877 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-terminal tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4877)
  AUTHORS   Yesbolatova A, Natsume T, Hayashi KI, Kanemaki MT
  TITLE     Generation of conditional auxin-inducible degron (AID) cells and 
            tight control of degron-fused proteins using the degradation 
            inhibitor auxinole.
  JOURNAL   Methods. 2019 Apr 23. pii: S1046-2023(18)30331-1. doi: 
            10.1016/j.ymeth.2019.04.010.
  PUBMED    31026591
REFERENCE   2  (bases 1 to 4877)
  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"; journalName: "Methods. 
            2019 Apr 23. pii: S1046-2023(18)30331-1. doi: 
            10.1016/j.ymeth.2019.04.010."
COMMENT     Sequence Label: pMK390(Stag-3FLAG-BSD)
FEATURES             Location/Qualifiers
     source          1..4877
                     /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"
     regulatory      1700..1709
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1706..2104
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     protein_bind    2607..2640
                     /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(2689..2709)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2689..2707)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2728..2744)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2728..2744)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2741..2763)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2752..2768
                     /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(2776..2782,2783..2800,2801..2806))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2821..2842
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2959..2976)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3130..3718)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3210..3229)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3889..4680,4681..4749))
                     /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     4512..4531
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4750..4854)
                     /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 ccaccatggc caagcctttg tctcaagaag aatccaccct
     1741 cattgaaaga gcaacggcta caatcaacag catccccatc tctgaagact acagcgtcgc
     1801 cagcgcagct ctctctagcg acggccgcat cttcactggt gtcaatgtat atcattttac
     1861 tgggggacct tgtgcagaac tcgtggtgct gggcactgct gctgctgcgg cagctggcaa
     1921 cctgacttgt atcgtcgcga tcggaaatga gaacaggggc atcttgagcc cctgcggacg
     1981 gtgccgacag gtgcttctcg atctgcatcc tgggatcaaa gccatagtga aggacagtga
     2041 tggacagccg acggcagttg ggattcgtga attgctgccc tctggttatg tgtgggaggg
     2101 ctaatcgatg atatcagatc cccgggatgc agaaattgat gatctattaa acaataaaga
     2161 tgtccactaa aatggaagtt tttcctgtca tactttgtta agaagggtga gaacagagta
     2221 cctacatttt gaatggaagg attggagcta cgggggtggg ggtggggtgg gattagataa
     2281 atgcctgctc tttactgaag gctctttact attgctttat gataatgttt catagttgga
     2341 tatcataatt taaacaagca aaaccaaatt aagggccagc tcattcctcc cactcatgat
     2401 ctatagatct atagatctct cgtgggatca ttgtttttct cttgattccc actttgtggt
     2461 tctaagtact gtggtttcca aatgtgtcag tttcatagcc tgaagaacga gatcagcagc
     2521 ctctgttcca catacacttc attctcagta ttgttttgcc aagttctaat tccatcagaa
     2581 gctggtcgag atccggaacc cttaatataa cttcgtataa tgtatgctat acgaagttat
     2641 taggtccctc gaagaggttc actaggatcc ggtacccagc ttttgttccc tttagtgagg
     2701 gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     2761 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     2821 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     2881 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     2941 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     3001 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     3061 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     3121 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     3181 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     3241 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     3301 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     3361 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     3421 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     3481 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     3541 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     3601 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     3661 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     3721 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     3781 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     3841 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     3901 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     3961 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     4021 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     4081 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     4141 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     4201 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     4261 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     4321 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     4381 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     4441 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     4501 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     4561 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     4621 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     4681 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     4741 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     4801 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     4861 tccccgaaaa gtgccac
//
