LOCUS       sequence-494849-        6840 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP-SUMO-Ser-mCherry N-degron reporter driven by aTc 
            responsive promoter, from KanR integrating mycobacterial plasmid 
            lacking Ulp1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6840)
  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 6840)
  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: pTKI-Ser
FEATURES             Location/Qualifiers
     source          1..6840
                     /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"
     rep_origin      complement(2127..2715)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2207..2226)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      2834..2861
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             3252..3875
                     /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(3325..3344)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(5878..5897)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5889..6704
                     /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(5962..5981)
                     /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 agcctgcgcg 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 gccttaaaaa aaaaagcccc gcgattgcgg ggcttttttt ttttaatacg gttatccaca
     2041 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     2101 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     2161 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     2221 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     2281 ctgtccgcct ttctcccctc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     2341 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     2401 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     2461 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     2521 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
     2581 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     2641 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     2701 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcatggaaca
     2761 ataaacgaaa ggctcagtcg aaagactggg cctttcgttt atctgttgtt tgaaagccgc
     2821 tgattagcta agcagaaggc catcctgacg gatggccttt ttctagaaaa aaaaaaaagc
     2881 gccgcaactg cggcgctttt ttttttttaa ttaaagaaat attggatcgt cggcaccgtc
     2941 acggccgtgg gaggcggcac gatccgcgac gtgatgatcg gccgcatccc cacggtgctg
     3001 cgcagtgagc tctacgccat cccggcgttg atctgtgcgt tcgcacgcac aggcccggtg
     3061 tgagaagggt ctctgacgag cgggagaacc cacccggggt gggcgagttt gtcctgcgtg
     3121 tgctcggtcg agtaggctct gggagtaccc gtgtgtacga ccagcacggc atacatcatt
     3181 tcgacgccga gagattcgcc gcccgaaatg agcacgatcc gcatggtacc atcaggagga
     3241 atcacttcgc aatgtctaga ttagataaaa gtaaagtgat taacagcgca ttagagctgc
     3301 ttaatgaggt cggaatcgaa ggtttaacaa cccgtaaact cgcccagaag cttggtgtag
     3361 agcagcctac attgtattgg catgtaaaaa ataagcgggc cctgctcgac gccttagcca
     3421 ttgagatgtt agataggcac catactcact tttgcccttt agaaggggaa agctggcaag
     3481 attttttacg taataacgct aaaagtttta gatgtgcttt actaagtcat cgcgatggag
     3541 caaaagtaca tttaggtaca cggcctacag aaaaacagta tgaaactctc gaaaatcaat
     3601 tagccttttt atgccaacaa ggtttttcac tagagaacgc gttatatgca ctcagcgctg
     3661 tggggcattt taccttaggt tgcgtattgg aagatcaaga gcatcaagtc gctaaagaag
     3721 aaagggaaac acctactact gatagtatgc cgccattatt acgacaagct atcgaattat
     3781 ttgatcacca aggtgcagag ccagccttct tattcggcct tgaattgatc atctgcggat
     3841 tagaaaaaca acttaaatgt gaaagtgggt cttaatgacc atgggctagc gctttagcta
     3901 attaatttaa aataaaaaag gggacctcta gggtccccaa ttaaatagtt aacactagta
     3961 ttaaaaaaaa aagccccgcg attgcggggc tttttttttt cgacggcgag ggttccgacc
     4021 gctgcaactc ccggtgcaac cttgtcccgg tctattctct tcactgcacc agctccaatc
     4081 tggtgtgaat gcccctcgtc tgttcgcgca ggcggggggc tctattcgtt tgtcagcatc
     4141 gaaagtagcc agatcaggga tgcgttgcaa ccgcgtatgc ccaggtcaga agagtcgcac
     4201 aagagttgca gacccctgga aagaaaaatg gccagagggc gaaaacaccc tctgaccagc
     4261 ggagcgggcg acgggaatcg aacccgcgta gctagtttgg aagaatgggt gtctgccgac
     4321 cacatatatg ggccggtcaa gataggtttt taccccctct cggctgcatc ctctaagtgg
     4381 aaagaaattg caggtcgtag aagcgcgttg aagcctgaga gttgcacagg agttgcaacc
     4441 cggtagcctt gttcacgacg agaggagacc tagttggcac gtcgcggatg gggatcgctg
     4501 aagactcagc gcagcgggag gatccaagcc tcatacgtca acccgcagga cggtgtgagg
     4561 tactacgcgc tgcagaccta cgacaacaag atggacgccg aagcctggct cgcgggcgag
     4621 aagcggctca tcgagatgga gacctggacc cctccacagg accgggcgaa gaaggcagcc
     4681 gccagcgcca tcacgctgga ggagtacacc cggaagtggc tcgtggagcg cgacctcgca
     4741 gacggcacca gggatctgta cagcgggcac gcggagcgcc gcatctaccc ggtgctaggt
     4801 gaagtggcgg tcacagagat gacgccagct ctggtgcgtg cgtggtgggc cgggatgggt
     4861 aggaagcacc cgactgcccg ccggcatgcc tacaacgtcc tccgggcggt gatgaacaca
     4921 gcggtcgagg acaagctgat cgcagagaac ccgtgccgga tcgagcagaa ggcagccgat
     4981 gagcgcgacg tagaggcgct gacgcctgag gagctggaca tcgtcgccgc tgagatcttc
     5041 gtgcactacc ggatcgcggc atacatcctg gcgtggacga gcctccggtt cggagagctg
     5101 atcgagcttc gccgcaagga catcgtggac gacggcatga cgatgaagct ccgggtgcgc
     5161 cgtggcgctt cccgcgtggg gaacaagatc gtcgttggca acgccaagac cgtccggtcg
     5221 aagcgtcctg tgacggttcc gcctcacgtc gcggagatga tccgagcgca catgaaggac
     5281 cgtacgaaga tgaacaaggg ccccgaggca ttcctggtga ccacgacgca gggcaaccgg
     5341 ctgtcgaagt ccgcgttcac caagtcgctg aagcgtggct acgccaagat cggtcggccg
     5401 gaactccgca tccacgacct ccgcgctgtc ggcgctacgt tcgccgctca ggcaggtgcg
     5461 acgaccaagg agctgatggc ccgcctcggt cacacgactc ctaggatggc gatgaagtac
     5521 cagatggcgt ctgaggcccg cgacgaggct atcgctgagg cgatgtccaa gctggccaag
     5581 acctcctgaa acgcaaaaag cccccctccc aaggacactg agtcctaaag aggggggttt
     5641 cttgtcagta cgcgaagaac cacgcctggc cgcgagcgcc agcaccgccg ctctgtgcgg
     5701 agacctgggc accagccccg ccgccgccag gagcattgcc gttcccgcca gctagccacc
     5761 tgacgtcggg ggggggggaa agccacgttg tgtctcaaaa tctctgatgt tacattgcac
     5821 aagataaaaa tatatcatca tgaacaataa aactgtctgc ttacataaac agtaatacaa
     5881 ggggtgttat gagccatatt caacgggaaa cgtcttgctc gaggccgcga ttaaattcca
     5941 acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg
     6001 cgacaatcta tcgcttgtat gggaagcccc atgcgccaga gttgtttctg aaacatggca
     6061 aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat
     6121 ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca
     6181 ccactgcgat ccccgggaaa acagcattcc aggtattaga agaatatcct gattcaggtg
     6241 aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta
     6301 attgtccttt taacagcgat cgcgtatttc gcctcgctca ggcgcaatca cgaatgaata
     6361 acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag
     6421 tctggaaaga aatgcataat cttttgccat tctcaccgga ttcagtcgtc actcatggtg
     6481 atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg
     6541 gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg
     6601 agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata
     6661 tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaatcagaa ttggttaatt
     6721 ggttgtaaca ctggcagagc attacgctga cttgacggga cggcggcttt gttgaataaa
     6781 tcgaactttt gctgagttga aggatcagat cacgcatctt cccgacaacg cagaccgttc
//
