LOCUS       sequence-235298-        4189 bp    DNA     circular SYN 14-APR-2025
DEFINITION  N-terminal tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4189)
  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 4189)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: pMK375 (Hygro-P2A-Stag-3FLAG)
FEATURES             Location/Qualifiers
     source          1..4189
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     68..90
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     82..99
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     83..99
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     109..128
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        109..127
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     152..168
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     153..169
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             190..1212
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             190..1212
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             1225..1281
                     /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             1288..1332
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             1345..1410
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(1430..1446)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1430..1446)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1483..1503)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1483..1501)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1522..1538)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1522..1538)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1535..1557)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1546..1562
                     /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(1570..1576,1577..1594,1595..1600))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1615..1636
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1753..1770)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1924..2512)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2004..2023)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2683..3474,3475..3543))
                     /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     3306..3325
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3544..3648)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3675..4131
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3762..3781)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3972..3993
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca
       61 gggttttccc agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta atacgactca
      121 ctatagggcg aattgggtac cgggcccccc ctcgaggtcg acggtatcga taagcttgat
      181 atcgccacca tgaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa
      241 aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc
      301 agcttcgatg taggagggcg tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc
      361 tacaaagatc gttatgttta tcggcacttt gcatcggccg cgctcccgat tccggaagtg
      421 cttgacattg gggaattcag cgagagcctg acctattgca tctcccgccg tgcacagggt
      481 gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag
      541 gccatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga
      601 ccgcaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc
      661 catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct
      721 ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg
      781 gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg
      841 agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg
      901 tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca
      961 ggatcgccgc ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc
     1021 ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc
     1081 cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg
     1141 accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg
     1201 agggcaaagg aatcaggaag cggagctact aacttcagcc tgctgaagca ggctggagac
     1261 gtggaggaga accctggacc tagatctaag gaaaccgccg ctgcaaaatt cgaacgccag
     1321 cacatggaca gccgatcccg ggctgactac aaagaccatg acggtgatta taaagatcat
     1381 gacatcgact acaaggatga cgatgacaag ggcgctggtg caggcgccgg atccactagt
     1441 tctagagcgg ccgccaccgc ggtggagctc cagcttttgt tccctttagt gagggttaat
     1501 tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     1561 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     1621 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     1681 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     1741 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     1801 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     1861 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     1921 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     1981 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     2041 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     2101 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     2161 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     2221 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     2281 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     2341 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     2401 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     2461 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     2521 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     2581 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     2641 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     2701 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     2761 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     2821 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     2881 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     2941 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     3001 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     3061 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     3121 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     3181 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     3241 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     3301 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     3361 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     3421 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     3481 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     3541 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     3601 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     3661 aaaagtgcca cctgacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     3721 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     3781 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     3841 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     3901 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     3961 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     4021 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa aatgagctga
     4081 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt tccattcgcc
     4141 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcg
//
