LOCUS       sequence-235303-        4993 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-terminal tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4993)
  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 4993)
  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: pMK345 (Hygro-P2A-mAID-mClover)
FEATURES             Location/Qualifiers
     source          1..4993
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     144..166
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     158..175
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     159..175
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     185..204
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        185..203
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     228..244
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     229..245
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             266..1288
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             266..1288
                     /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             1301..1357
                     /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             1364..1567
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Kubota et al., 2013)"
                     /label=mini-AID
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEA
                     AAFVKVSMDGAPYLRKIDLRMYK"
     CDS             join(1574..1576,1577..1579,1580..2290)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(1619..1640)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2227..2248
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2310..2326)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2310..2326)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2363..2383)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2363..2381)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2402..2418)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2402..2418)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2415..2437)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2426..2442
                     /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(2450..2456,2457..2474,2475..2480))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2495..2516
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2633..2650)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2804..3392)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2884..2903)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3563..4354,4355..4423))
                     /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     4186..4205
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4424..4528)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4555..4984
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4642..4661)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4852..4873
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 tattaacgct tacaatttcc attcgccatt caggctgcgc aactgttggg aagggcgatc
       61 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
      121 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg
      181 cgcgtaatac gactcactat agggcgaatt gggtaccggg ccccccctcg aggtcgacgg
      241 tatcgataag cttgatatcg ccaccatgaa aaagcctgaa ctcaccgcga cgtctgtcga
      301 gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga
      361 agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag
      421 ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct
      481 cccgattccg gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc
      541 ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct
      601 gcagccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg
      661 gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg
      721 cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc
      781 gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg
      841 gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac
      901 agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat
      961 cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag
     1021 gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga
     1081 ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg
     1141 atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag
     1201 aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg
     1261 ccccagcact cgtccgaggg caaaggaatc aggaagcgga gctactaact tcagcctgct
     1321 gaagcaggct ggagacgtgg aggagaaccc tggacctaga tctaaggaga agagtgcttg
     1381 tcctaaagat ccagccaaac ctccggccaa ggcacaagtt gtgggatggc caccggtgag
     1441 atcataccgg aagaacgtga tggtttcctg ccaaaaatca agcggtggcc cggaggcggc
     1501 ggcgttcgtg aaggtatcaa tggacggagc accgtacttg aggaaaatcg atttgaggat
     1561 gtataaagct agcatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     1621 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtccgcggcg agggcgaggg
     1681 cgatgccacc aacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     1741 gccctggccc accctcgtga ccaccttcgg ctacggcgtg gcctgcttca gccgctaccc
     1801 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     1861 gcgcaccatc tctttcaagg acgacggtac ctacaagacc cgcgccgagg tgaagttcga
     1921 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     1981 catcctgggg cacaagctgg agtacaactt caacagccac aacgtctata tcacggccga
     2041 caagcagaag aacggcatca aggctaactt caagatccgc cacaacgttg aggacggcag
     2101 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     2161 gcccgacaac cactacctga gccatcagtc caagctgagc aaagacccca acgagaagcg
     2221 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg attacacatg gcatggacga
     2281 gctgtacaag ggcgctggtg caggcgccgg atccactagt tctagagcgg ccgccaccgc
     2341 ggtggagctc cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat
     2401 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     2461 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     2521 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     2581 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     2641 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     2701 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     2761 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     2821 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     2881 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     2941 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     3001 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     3061 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     3121 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     3181 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     3241 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     3301 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     3361 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     3421 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     3481 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     3541 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     3601 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     3661 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     3721 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     3781 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     3841 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     3901 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     3961 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     4021 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     4081 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     4141 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     4201 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     4261 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     4321 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     4381 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     4441 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     4501 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgcgc
     4561 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     4621 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     4681 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     4741 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     4801 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     4861 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     4921 ttttgccgat ttcggcctat tggttaaaaa aatgagctga tttaacaaaa atttaacgcg
     4981 aattttaaca aaa
//
