LOCUS       sequence-331861-        4040 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 4040)
  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 4040)
  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: pMK439 (Stag-3FLAG-P2A-BSD)
FEATURES             Location/Qualifiers
     source          1..4040
                     /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             863..1261
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     protein_bind    1764..1797
                     /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(1846..1866)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1846..1864)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1885..1901)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1885..1901)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1898..1920)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1909..1925
                     /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(1933..1939,1940..1957,1958..1963))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1978..1999
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2116..2133)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2287..2875)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2367..2386)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3046..3837,3838..3906))
                     /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     3669..3688
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3907..4011)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(4038..4040,1..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 agatggccaa gcctttgtct caagaagaat ccaccctcat
      901 tgaaagagca acggctacaa tcaacagcat ccccatctct gaagactaca gcgtcgccag
      961 cgcagctctc tctagcgacg gccgcatctt cactggtgtc aatgtatatc attttactgg
     1021 gggaccttgt gcagaactcg tggtgctggg cactgctgct gctgcggcag ctggcaacct
     1081 gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc ttgagcccct gcggacggtg
     1141 ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc atagtgaagg acagtgatgg
     1201 acagccgacg gcagttggga ttcgtgaatt gctgccctct ggttatgtgt gggagggcta
     1261 atcgatgata tcagatcccc gggatgcaga aattgatgat ctattaaaca ataaagatgt
     1321 ccactaaaat ggaagttttt cctgtcatac tttgttaaga agggtgagaa cagagtacct
     1381 acattttgaa tggaaggatt ggagctacgg gggtgggggt ggggtgggat tagataaatg
     1441 cctgctcttt actgaaggct ctttactatt gctttatgat aatgtttcat agttggatat
     1501 cataatttaa acaagcaaaa ccaaattaag ggccagctca ttcctcccac tcatgatcta
     1561 tagatctata gatctctcgt gggatcattg tttttctctt gattcccact ttgtggttct
     1621 aagtactgtg gtttccaaat gtgtcagttt catagcctga agaacgagat cagcagcctc
     1681 tgttccacat acacttcatt ctcagtattg ttttgccaag ttctaattcc atcagaagct
     1741 ggtcgagatc cggaaccctt aatataactt cgtataatgt atgctatacg aagttattag
     1801 gtccctcgaa gaggttcact aggatccggt acccagcttt tgttcccttt agtgagggtt
     1861 aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     1921 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     1981 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     2041 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     2101 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     2161 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     2221 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     2281 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     2341 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     2401 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     2461 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     2521 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     2581 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     2641 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     2701 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     2761 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     2821 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     2881 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     2941 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     3001 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     3061 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     3121 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     3181 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     3241 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     3301 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     3361 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     3421 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     3481 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     3541 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     3601 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     3661 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     3721 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     3781 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     3841 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     3901 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     3961 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     4021 ccgaaaagtg ccacctgacg
//
