LOCUS       sequence-234565-        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"
     primer_bind     102..124
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     116..133
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     117..133
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     143..162
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        143..161
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             194..238
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             251..316
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    448..569
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(485..504)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     539..558
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    600..633
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        694..1193
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(729..752)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1126..1145
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      1217..1226
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1223..1621
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     protein_bind    2124..2157
                     /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(2206..2226)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2206..2224)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2245..2261)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2245..2261)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2258..2280)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2269..2285
                     /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(2293..2299,2300..2317,2318..2323))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2338..2359
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2476..2493)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2647..3235)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2727..2746)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3406..4197,4198..4266))
                     /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     4029..4048
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4267..4371)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4398..4853
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4485..4504)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4695..4716
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
       61 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa
      121 aacgacggcc agtgagcgcg cgtaatacga ctcactatag ggcgaattgg agctcggatc
      181 cggtgcaggc gccaaggaaa ccgccgctgc aaaattcgaa cgccagcaca tggacagccg
      241 atcccgggct gactacaaag accatgacgg tgattataaa gatcatgaca tcgactacaa
      301 ggatgacgat gacaagtagt gtacaagtaa tctagataac tgatcataat cagccatacc
      361 acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa
      421 cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa
      481 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
      541 ggtttgtcca aactcatcaa tgtatcttaa cgcgtcgatc atattcaata acccttaata
      601 taacttcgta taatgtatgc tatacgaagt tattaggtct gaagaggagt ttacgtccag
      661 ccaagcttag gatctcgacc tcgaaattct accgggtagg ggaggcgctt ttcccaaggc
      721 agtctggagc atgcgcttta gcagccccgc tgggcacttg gcgctacaca agtggcctct
      781 ggcctcgcac acattccaca tccaccggta ggcgccaacc gactccgttc tttggtggcc
      841 ccttcgcgcc accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg
      901 cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac tagtctcgtg cagatggaca
      961 gcaccgctga gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct
     1021 ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg
     1081 ggctcagggg cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc
     1141 aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc tccgggcctt tcgacctgca
     1201 tccatctaga tctcgagcca ccatggccaa gcctttgtct caagaagaat ccaccctcat
     1261 tgaaagagca acggctacaa tcaacagcat ccccatctct gaagactaca gcgtcgccag
     1321 cgcagctctc tctagcgacg gccgcatctt cactggtgtc aatgtatatc attttactgg
     1381 gggaccttgt gcagaactcg tggtgctggg cactgctgct gctgcggcag ctggcaacct
     1441 gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc ttgagcccct gcggacggtg
     1501 ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc atagtgaagg acagtgatgg
     1561 acagccgacg gcagttggga ttcgtgaatt gctgccctct ggttatgtgt gggagggcta
     1621 atcgatgata tcagatcccc gggatgcaga aattgatgat ctattaaaca ataaagatgt
     1681 ccactaaaat ggaagttttt cctgtcatac tttgttaaga agggtgagaa cagagtacct
     1741 acattttgaa tggaaggatt ggagctacgg gggtgggggt ggggtgggat tagataaatg
     1801 cctgctcttt actgaaggct ctttactatt gctttatgat aatgtttcat agttggatat
     1861 cataatttaa acaagcaaaa ccaaattaag ggccagctca ttcctcccac tcatgatcta
     1921 tagatctata gatctctcgt gggatcattg tttttctctt gattcccact ttgtggttct
     1981 aagtactgtg gtttccaaat gtgtcagttt catagcctga agaacgagat cagcagcctc
     2041 tgttccacat acacttcatt ctcagtattg ttttgccaag ttctaattcc atcagaagct
     2101 ggtcgagatc cggaaccctt aatataactt cgtataatgt atgctatacg aagttattag
     2161 gtccctcgaa gaggttcact aggatccggt acccagcttt tgttcccttt agtgagggtt
     2221 aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     2281 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     2341 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     2401 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     2461 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     2521 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     2581 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     2641 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     2701 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     2761 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     2821 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     2881 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     2941 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     3001 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     3061 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     3121 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     3181 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     3241 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     3301 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     3361 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     3421 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     3481 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     3541 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     3601 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     3661 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     3721 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     3781 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     3841 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     3901 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     3961 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     4021 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     4081 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     4141 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     4201 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     4261 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     4321 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     4381 ccgaaaagtg ccacctgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     4441 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     4501 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     4561 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     4621 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     4681 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     4741 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     4801 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttccattcg
     4861 ccattcaggc tgcgcaa
//
