LOCUS       sequence-494847-        7734 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP-SUMO-Tyr-mCherry N-degron reporter driven by aTc 
            responsive promoter, and constitutively expresses Ulp1, from KanR 
            integrating mycobacterial plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7734)
  AUTHORS   Presloid CJ, Jiang J, Kandel P, Anderson HR, Beardslee PC, Swayne 
            TM, Schmitz KR
  TITLE     ClpS Directs Degradation of N-Degron Substrates With Primary 
            Destabilizing Residues in Mycolicibacterium smegmatis.
  JOURNAL   Mol Microbiol. 2025 Jan;123(1):16-30. doi: 10.1111/mmi.15334. Epub 
            2024 Dec 3.
  PUBMED    39626090
REFERENCE   2  (bases 1 to 7734)
  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"; doi: "10.1111/mmi.15334";
            journalName: "Mol Microbiol"; date: "2025-01"; volume: "123"; issue:
            "1"; pages: "16-30"
COMMENT     Sequence Label: pTKIU-Tyr
FEATURES             Location/Qualifiers
     source          1..7734
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             163..189
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             190..900
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="RKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             1942..1971
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2800..2865
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     rep_origin      complement(3021..3609)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3101..3120)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      3728..3755
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             4146..4769
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(4219..4238)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(6772..6791)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6783..7598
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPHAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHNLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(6856..6875)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 cgtggcaaag caaaagttca aaatcaccaa ctggtccacc tacaacaaag ctctcaccaa
       61 ccgtggtctg accagggaaa atagccctct gacctgggga tttgcgatcc ctatcagtga
      121 tagatataat ctgggagtac tgactcaggc ggtatccata tgtacccgta cgacgttccg
      181 gactacgccc gtaaaggcga agagctgttc actggtgtcg tccctattct ggtggaactg
      241 gatggtgatg tcaacggtca taagttttcc gtgcgtggcg agggtgaagg tgacgcaact
      301 aatggtaaac tgacgctgaa gttcatctgt actactggta aactgccggt accttggccg
      361 actctggtaa cgacgctgac ttatggtgtt cagtgctttg ctcgttatcc ggaccatatg
      421 aagcagcatg acttcttcaa gtccgccatg ccggaaggct atgtgcagga acgcacgatt
      481 tcctttaagg atgacggcac gtacaaaacg cgtgcggaag tgaaatttga aggcgatacc
      541 ctggtaaacc gcattgagct gaaaggcatt gactttaaag aagacggcaa tatcctgggc
      601 cataagctgg aatacaattt taacagccac aatgtttaca tcaccgccga taaacaaaaa
      661 aatggcatta aagcgaattt taaaattcgc cacaacgtgg aggatggcag cgtgcagctg
      721 gctgatcact accagcaaaa cactccaatc ggtgatggtc ctgttctgct gccagacaat
      781 cactatctga gcacgcaaag cgttctgtct aaagatccga acgagaaacg cgatcatatg
      841 gttctgctgg agttcgtaac cgcagcgggc atcacgcatg gcatggatga actgtacaaa
      901 ggccaattga tggactcgga ggtcaaccag gaggccaagc ccgaggtcaa accggaagtg
      961 aagccggaga cccacatcaa cctgaaagtg agcgacggta gctcggagat cttcttcaag
     1021 atcaagaaga ccaccccgct gcgccggctg atggaagcct tcgcgaagcg ccaaggcaaa
     1081 gagatggact cgctccgctt cctctacgac ggtatccgca tccaggccga ccaaaccccc
     1141 gaggatctgg acatggagga caatgacatc atcgaagctt accgcgagca gatcggtggc
     1201 tacctgcgcg tccaatctgg cacagccagc ctgcaggtct cgaaggggga agaggacaac
     1261 atggccatca tcaaggaatt catgcggttc aaggtgcaca tggagggcag cgtcaatggg
     1321 catgagttcg agatcgaggg ggaaggtgag gggcggccgt acgaaggcac ccagacggcc
     1381 aagctcaagg tcacgaaggg cggcccgctc ccgttcgcct gggacatcct gagcccgcag
     1441 ttcatgtacg gctcgaaggc ctacgtgaag catccggccg acatcccgga ctacctcaag
     1501 ctctcgttcc ccgaaggctt caagtgggag cgtgtcatga acttcgagga cggcggtgtg
     1561 gtcacggtca cccaggactc ctcgctgcaa gatggggaat tcatctacaa ggtcaagctc
     1621 cgggggacca acttcccctc ggacggtccg gtgatgcaga agaagaccat gggttgggaa
     1681 gccagctccg agcgcatgta cccggaggac ggtgccctga agggggaaat caagcagcgg
     1741 ctcaagctga aggacggtgg tcattacgac gccgaggtga agaccaccta caaggccaaa
     1801 aagccggtcc aactgcccgg cgcctacaac gtgaacatca aactggatat cacctcgcac
     1861 aacgaggact acaccatcgt cgagcagtac gagcgcgccg agggtcgcca cagcaccggt
     1921 ggcatggatg aactgtacaa agaacaaaaa ctcatctcag aagaggatct gtaagcggcc
     1981 gccttaagaa aggaggaagg atttttcatg cacgcaaccc ctgaggagag tattcggatc
     2041 atgtccgggc tccggccgcc cgcccggcag ctgatcaccc ccctcagcga ggaatggcgg
     2101 tcccgggtgg aggcggcccg gaatgccaac ccggcgacgg aactcgcgaa gacgctggag
     2161 ggccagccgc tggtgcgccg ggacttcgaa gaaaagctcc tgcccgccac cgcctggctg
     2221 aacgacaacg tcatcatcgg ggccatcttc tacatcgcgg actacgtgaa caccaagaaa
     2281 ggggcgccga accaggagcc caagtgcacc gccttcacgt cgttcttctg gccccggctg
     2341 ctctcccacg ggccgggcgg ctgcgggcgc ctcctgcgcc gcgcgaacgt gcggaaggcc
     2401 aacttcctgg atatcgacac catcctgatc ccgatctgcg aaagctcgca ctggaccctg
     2461 gcggtgatcc ggcccgggcg gcgcaccgtc tcgcacctcg actcgatggc cgcggggcgc
     2521 ggctccgagc gggtgaaggc gaagctcctc gaactggtca agttcgtgct ggaagaccag
     2581 ttcgtggagg ccgaatggca agcggtggac ttccaggcgc cccgccagac gaacggctgg
     2641 gactgcggag tcttcaccat caccaacgcg atctgtctgg cgctgggcgt ggacccggcc
     2701 caggcgtaca ccgaagccca gctgccccta cagcgccagc gcatcgcggc ggtcctgctc
     2761 aacggtggct tcaagggtga cttcaccctc gacgacctgg actacaagga ccacgatggt
     2821 gattataagg accacgacat cgactacaaa gacgacgacg ataagtaagc ggccgcctta
     2881 aaaaaaaaag ccccgcgatt gcggggcttt ttttttttaa tacggttatc cacagaatca
     2941 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     3001 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     3061 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     3121 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     3181 gcctttctcc cctcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     3241 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     3301 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     3361 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     3421 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     3481 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     3541 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     3601 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcatgg aacaataaac
     3661 gaaaggctca gtcgaaagac tgggcctttc gtttatctgt tgtttgaaag ccgctgatta
     3721 gctaagcaga aggccatcct gacggatggc ctttttctag aaaaaaaaaa aagcgccgca
     3781 actgcggcgc tttttttttt ttaattaaag aaatattgga tcgtcggcac cgtcacggcc
     3841 gtgggaggcg gcacgatccg cgacgtgatg atcggccgca tccccacggt gctgcgcagt
     3901 gagctctacg ccatcccggc gttgatctgt gcgttcgcac gcacaggccc ggtgtgagaa
     3961 gggtctctga cgagcgggag aacccacccg gggtgggcga gtttgtcctg cgtgtgctcg
     4021 gtcgagtagg ctctgggagt acccgtgtgt acgaccagca cggcatacat catttcgacg
     4081 ccgagagatt cgccgcccga aatgagcacg atccgcatgg taccatcagg aggaatcact
     4141 tcgcaatgtc tagattagat aaaagtaaag tgattaacag cgcattagag ctgcttaatg
     4201 aggtcggaat cgaaggttta acaacccgta aactcgccca gaagcttggt gtagagcagc
     4261 ctacattgta ttggcatgta aaaaataagc gggccctgct cgacgcctta gccattgaga
     4321 tgttagatag gcaccatact cacttttgcc ctttagaagg ggaaagctgg caagattttt
     4381 tacgtaataa cgctaaaagt tttagatgtg ctttactaag tcatcgcgat ggagcaaaag
     4441 tacatttagg tacacggcct acagaaaaac agtatgaaac tctcgaaaat caattagcct
     4501 ttttatgcca acaaggtttt tcactagaga acgcgttata tgcactcagc gctgtggggc
     4561 attttacctt aggttgcgta ttggaagatc aagagcatca agtcgctaaa gaagaaaggg
     4621 aaacacctac tactgatagt atgccgccat tattacgaca agctatcgaa ttatttgatc
     4681 accaaggtgc agagccagcc ttcttattcg gccttgaatt gatcatctgc ggattagaaa
     4741 aacaacttaa atgtgaaagt gggtcttaat gaccatgggc tagcgcttta gctaattaat
     4801 ttaaaataaa aaaggggacc tctagggtcc ccaattaaat agttaacact agtattaaaa
     4861 aaaaaagccc cgcgattgcg gggctttttt ttttcgacgg cgagggttcc gaccgctgca
     4921 actcccggtg caaccttgtc ccggtctatt ctcttcactg caccagctcc aatctggtgt
     4981 gaatgcccct cgtctgttcg cgcaggcggg gggctctatt cgtttgtcag catcgaaagt
     5041 agccagatca gggatgcgtt gcaaccgcgt atgcccaggt cagaagagtc gcacaagagt
     5101 tgcagacccc tggaaagaaa aatggccaga gggcgaaaac accctctgac cagcggagcg
     5161 ggcgacggga atcgaacccg cgtagctagt ttggaagaat gggtgtctgc cgaccacata
     5221 tatgggccgg tcaagatagg tttttacccc ctctcggctg catcctctaa gtggaaagaa
     5281 attgcaggtc gtagaagcgc gttgaagcct gagagttgca caggagttgc aacccggtag
     5341 ccttgttcac gacgagagga gacctagttg gcacgtcgcg gatggggatc gctgaagact
     5401 cagcgcagcg ggaggatcca agcctcatac gtcaacccgc aggacggtgt gaggtactac
     5461 gcgctgcaga cctacgacaa caagatggac gccgaagcct ggctcgcggg cgagaagcgg
     5521 ctcatcgaga tggagacctg gacccctcca caggaccggg cgaagaaggc agccgccagc
     5581 gccatcacgc tggaggagta cacccggaag tggctcgtgg agcgcgacct cgcagacggc
     5641 accagggatc tgtacagcgg gcacgcggag cgccgcatct acccggtgct aggtgaagtg
     5701 gcggtcacag agatgacgcc agctctggtg cgtgcgtggt gggccgggat gggtaggaag
     5761 cacccgactg cccgccggca tgcctacaac gtcctccggg cggtgatgaa cacagcggtc
     5821 gaggacaagc tgatcgcaga gaacccgtgc cggatcgagc agaaggcagc cgatgagcgc
     5881 gacgtagagg cgctgacgcc tgaggagctg gacatcgtcg ccgctgagat cttcgtgcac
     5941 taccggatcg cggcatacat cctggcgtgg acgagcctcc ggttcggaga gctgatcgag
     6001 cttcgccgca aggacatcgt ggacgacggc atgacgatga agctccgggt gcgccgtggc
     6061 gcttcccgcg tggggaacaa gatcgtcgtt ggcaacgcca agaccgtccg gtcgaagcgt
     6121 cctgtgacgg ttccgcctca cgtcgcggag atgatccgag cgcacatgaa ggaccgtacg
     6181 aagatgaaca agggccccga ggcattcctg gtgaccacga cgcagggcaa ccggctgtcg
     6241 aagtccgcgt tcaccaagtc gctgaagcgt ggctacgcca agatcggtcg gccggaactc
     6301 cgcatccacg acctccgcgc tgtcggcgct acgttcgccg ctcaggcagg tgcgacgacc
     6361 aaggagctga tggcccgcct cggtcacacg actcctagga tggcgatgaa gtaccagatg
     6421 gcgtctgagg cccgcgacga ggctatcgct gaggcgatgt ccaagctggc caagacctcc
     6481 tgaaacgcaa aaagcccccc tcccaaggac actgagtcct aaagaggggg gtttcttgtc
     6541 agtacgcgaa gaaccacgcc tggccgcgag cgccagcacc gccgctctgt gcggagacct
     6601 gggcaccagc cccgccgccg ccaggagcat tgccgttccc gccagctagc cacctgacgt
     6661 cggggggggg ggaaagccac gttgtgtctc aaaatctctg atgttacatt gcacaagata
     6721 aaaatatatc atcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg
     6781 ttatgagcca tattcaacgg gaaacgtctt gctcgaggcc gcgattaaat tccaacatgg
     6841 atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa
     6901 tctatcgctt gtatgggaag ccccatgcgc cagagttgtt tctgaaacat ggcaaaggta
     6961 gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc
     7021 ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg
     7081 cgatccccgg gaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata
     7141 ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc
     7201 cttttaacag cgatcgcgta tttcgcctcg ctcaggcgca atcacgaatg aataacggtt
     7261 tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga
     7321 aagaaatgca taatcttttg ccattctcac cggattcagt cgtcactcat ggtgatttct
     7381 cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag
     7441 tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt
     7501 ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata
     7561 aattgcagtt tcatttgatg ctcgatgagt ttttctaatc agaattggtt aattggttgt
     7621 aacactggca gagcattacg ctgacttgac gggacggcgg ctttgttgaa taaatcgaac
     7681 ttttgctgag ttgaaggatc agatcacgca tcttcccgac aacgcagacc gttc
//
