LOCUS       sequence-331860-        4736 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-terminal tagging with Stag-3FLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4736)
  AUTHORS   Saito Y, Kanemaki MT
  TITLE     Targeted Protein Depletion Using the Auxin-Inducible Degron 2 (AID2)
            System.
  JOURNAL   Curr Protoc. 2021 Aug;1(8):e219. doi: 10.1002/cpz1.219.
  PUBMED    34370399
REFERENCE   2  (bases 1 to 4736)
  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: "Curr 
            Protoc."; date: "2021-08"; volume: "1(8):e219. doi"; pages: " 
            10.1002/cpz1.219"
COMMENT     Sequence Label: pMK438 (Stag-3FLAG-P2A-Hygro)
FEATURES             Location/Qualifiers
     source          1..4736
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(85..104)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     295..316
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     579..601
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     593..610
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     594..610
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     620..639
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        620..638
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             671..715
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             728..793
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             800..856
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             878..1897
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             878..1897
                     /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    2460..2493
                     /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(2542..2562)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2542..2560)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2581..2597)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2581..2597)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2594..2616)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2605..2621
                     /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(2629..2635,2636..2653,2654..2659))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2674..2695
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2812..2829)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2983..3571)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3063..3082)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3742..4533,4534..4602))
                     /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     4365..4384
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4603..4707)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(4734..4736,1..453)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
       61 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
      121 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
      181 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
      241 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
      301 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
      361 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
      421 cgaattttaa caaaatatta acgcttacaa tttccattcg ccattcaggc tgcgcaactg
      481 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
      541 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac
      601 gacggccagt gagcgcgcgt aatacgactc actatagggc gaattggagc tcggatccgg
      661 tgcaggcgcc aaggaaaccg ccgctgcaaa attcgaacgc cagcacatgg acagccgatc
      721 ccgggctgac tacaaagacc atgacggtga ttataaagat catgacatcg actacaagga
      781 tgacgatgac aagacgcgtg ctactaactt cagcctgctg aagcaggctg gagacgtgga
      841 ggagaaccct ggacctctcg agatggatag atccggaaag cctgaactca ccgcgacgtc
      901 tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga
      961 gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt
     1021 aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc
     1081 cgcgctcccg attccggaag tgcttgacat tggggaattc agcgagagcc tgacctattg
     1141 catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc
     1201 tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac
     1261 gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt
     1321 catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt
     1381 cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga
     1441 agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg
     1501 cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc
     1561 caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga
     1621 gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg
     1681 tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca
     1741 gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc
     1801 ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa
     1861 ccgacgcccc agcactcgtc cgagggcaaa ggaataggct agccgcccgc cccacgaccc
     1921 gcagcgcccg accgaaagga gcgcacgacc ccatgcatcg atgatatcag atccccggga
     1981 tgcagaaatt gatgatctat taaacaataa agatgtccac taaaatggaa gtttttcctg
     2041 tcatactttg ttaagaaggg tgagaacaga gtacctacat tttgaatgga aggattggag
     2101 ctacgggggt gggggtgggg tgggattaga taaatgcctg ctctttactg aaggctcttt
     2161 actattgctt tatgataatg tttcatagtt ggatatcata atttaaacaa gcaaaaccaa
     2221 attaagggcc agctcattcc tcccactcat gatctataga tctatagatc tctcgtggga
     2281 tcattgtttt tctcttgatt cccactttgt ggttctaagt actgtggttt ccaaatgtgt
     2341 cagtttcata gcctgaagaa cgagatcagc agcctctgtt ccacatacac ttcattctca
     2401 gtattgtttt gccaagttct aattccatca gaagctggtc gagatccgga acccttaata
     2461 taacttcgta taatgtatgc tatacgaagt tattaggtcc ctcgaagagg ttcactagga
     2521 tccggtaccc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg
     2581 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     2641 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     2701 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     2761 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     2821 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     2881 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     2941 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     3001 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     3061 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     3121 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     3181 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     3241 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     3301 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     3361 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     3421 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     3481 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     3541 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     3601 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     3661 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     3721 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     3781 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     3841 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     3901 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     3961 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     4021 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     4081 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     4141 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     4201 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     4261 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     4321 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     4381 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     4441 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     4501 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     4561 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     4621 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     4681 gaaaaataaa caaatagagg ttccgcgcac atttccccga aaagtgccac ctgacg
//
