LOCUS       sequence-420124-        5653 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pkk223 with Marinosulfonomonas MutY containing D150N and E49Q 
            mutations to reduce catalytic activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5653)
  AUTHORS   Utzman PH, Mays VP, Miller BC, Fairbanks MC, Brazelton WJ, Horvath 
            MP
  TITLE     Metagenome mining and functional analysis reveal oxidized guanine 
            DNA repair at the Lost City Hydrothermal Field.
  JOURNAL   PLoS One. 2024 May 8;19(5):e0284642. doi: 
            10.1371/journal.pone.0284642. eCollection 2024.
  PUBMED    38718041
REFERENCE   2  (bases 1 to 5653)
  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: "PLoS One.";
            date: "2024-05-8"; volume: "19(5):e0284642. doi"; pages: " 
            10.1371/journal.pone.0284642. eCollection 2024"
COMMENT     Sequence Label: pkk223_MsMutY_D150N_E49Q
FEATURES             Location/Qualifiers
     source          1..5653
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1136..1153)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1306..1392
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1484..1511
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1530..1621
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1622..1690,1691..2482)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1840..1859)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2653..3241
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3142..3161
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3395..3412
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3427..3567
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3493..3512)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3653..3675
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3669..3860)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5318..5337
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(5584..5589,5590..5605,5606..5612)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    5620..5636
                     /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)."
     primer_bind     5625..5647
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 aattcggtga ccatgcgcga ctaccgcgac aactccgaac tggggaccgt ccttctgggg
       61 tggtacgatg ctaacgcgag aagcttaccc tggcgtgtag ccccagcacg ttcgaaagca
      121 ggggctttgc cggatccata ccgtatttgg atgagccaag tcatgctgca gcaaacgact
      181 gtcgctgcag tcgctaaata ttttcacgcg tttacgactc gctggccaac cattatcgcc
      241 ttggcatctg ccaatgacga tgatgttatg ggacagtggg caggcctggg ttactatgct
      301 cgtgcacgca accttttgaa gtgtgcacgc gtggtagtag cggattatgg cggcaatttc
      361 cccgacacac gcgatgagtt gcttaaactt cctggcattg gaccctatac ggcggcagct
      421 attgcaagta tcgccttcgg tcgttcagaa acagtgatga atgcgaatat cgaacgcgtg
      481 atggcacgta tgcatgacat ccattcgccg ttgcccagtg ctaagcctga acttcttgca
      541 tgcgcggcag cccttacccc ggatttccgt gccggcgatt atgctcaagc tgtaatggac
      601 ttaggcgcca ccatttgcac gccgaagtct gcatcttgtg gagtgtgccc ctggtccttc
      661 gcatgcctag cccgtatcaa tggtacagct gccgtcttac cgaaaaagac tcctaaaccc
      721 gtcaagccaa cacgctacgg aatcgcgtac atcgcaagac gtggggacgg tgcgtggttg
      781 ttagaacgcc gtcctgataa aggtttactg ggaggaatgc tgggctggcc cggggctcag
      841 tggggcgatc aggcggtaga caagccgcct gtttcagctg attggcaaga ccccggtgcc
      901 gaagtgcgcc attcatttac gcactttcat ctgcgtttat cgttacgcat tagcgtcgtc
      961 ggtaatgcga aaccaaccgc agggcacttc atcgcggatg cagattttga ccccgagaca
     1021 ctacccaccg taatgcgtaa agcatatcgt attgcacgca caaagtttga cggcgacgcg
     1081 aacggttgac tgcagccaag cttggctgtt ttggcggatg agagaagatt ttcagcctga
     1141 tacagattaa atcagaacgc agaagcggtc tgataaaaca gaatttgcct ggcggcagta
     1201 gcgcggtggt cccacctgac cccatgccga actcagaagt gaaacgccgt agcgccgatg
     1261 gtagtgtggg gtctccccat gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag
     1321 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
     1381 agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg
     1441 cgggcaggac gcccgccata aactgccagg catcaaatta agcagaaggc catcctgacg
     1501 gatggccttt ttgcgtttct acaaactctt ttgtttattt ttctaaatac attcaaatat
     1561 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     1621 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     1681 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     1741 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     1801 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     1861 ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     1921 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     1981 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     2041 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     2101 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     2161 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     2221 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     2281 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     2341 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     2401 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     2461 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     2521 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     2581 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     2641 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     2701 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     2761 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     2821 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     2881 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     2941 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3001 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     3061 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     3121 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     3181 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     3241 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     3301 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     3361 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     3421 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg
     3481 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     3541 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     3601 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     3661 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc
     3721 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg
     3781 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg
     3841 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta
     3901 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
     3961 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
     4021 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt
     4081 agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc
     4141 gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca
     4201 gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa
     4261 ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc
     4321 acccgtggcc aggacccaac gctgcccgag atgcgccgcg tgcggctgct ggagatggcg
     4381 gacgcgatgg atatgttctg ccaagggttg gtttgcgcat tcacagttct ccgcaagaat
     4441 tgattggctc caattcttgg agtggtgaat ccgttagcga ggtgccgccg gcttccattc
     4501 aggtcgaggt ggcccggctc catgcaccgc gacgcaacgc ggggaggcag acaaggtata
     4561 gggcggcgcc tacaatccat gccaacccgt tccatgtgct cgccgaggcg gcataaatcg
     4621 ccgtgacgat cagcggtcca atgatcgaag ttaggctggt aagagccgcg agcgatcctt
     4681 gaagctgtcc ctgatggtcg tcatctacct gcctggacag catggcctgc aacgcgggca
     4741 tcccgatgcc gccggaagcg agaagaatca taatggggaa ggccatccag cctcgcgtcg
     4801 cgaacgccag caagacgtag cccagcgcgt cggccgccat gccggcgata atggcctgct
     4861 tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga
     4921 ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc
     4981 cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag
     5041 tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga
     5101 aggctctcaa gggcatcggt cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     5161 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     5221 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     5281 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     5341 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcc
     5401 gggcttatcg actgcacggt gcaccaatgc ttctggcgtc aggcagccat cggaagctgt
     5461 ggtatggctg tgcaggtcgt aaatcactgc ataattcgtg tcgctcaagg cgcactcccg
     5521 ttctggataa tgttttttgc gccgacatca taacggttct ggcaaatatt ctgaaatgag
     5581 ctgttgacaa ttaatcatcg gctcgtataa tgtgtggaat tgtgagcgga taacaatttc
     5641 acacaggaaa cag
//
