LOCUS       sequence-420121-        5689 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pkk223 with Thiotrichaceae MutY containing D147N and E46Q mutations 
            to reduce catalytic activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5689)
  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 5689)
  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_TtMutY_D147N_E46Q
FEATURES             Location/Qualifiers
     source          1..5689
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1175..1192)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1345..1431
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1523..1550
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1569..1660
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1661..1729,1730..2521)
                     /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(1879..1898)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2692..3280
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3181..3200
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3434..3451
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3466..3606
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3532..3551)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3692..3714
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3708..3899)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5357..5376
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(5623..5628,5629..5644,5645..5651)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    5659..5675
                     /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     5664..5686
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 cagaattcgg tgaccatggg tgcgagcaag tggcgcgtct ttgaagctgt catccaattt
       61 cccgagaaga tcctagtgtg gtttgacgtg cacgggcgta aggaccttcc gtggcaaaaa
      121 aatattaatc cgtaccgtgt ttgggtgtcg cagattatgc ttcagcaaac ccaggttaag
      181 acagttattc cctacttcga gaaattcatg gctactttcc cagacgttca aactttagct
      241 cacgctgacc aggacttagt attgaaacac tggtccggac tgggctacta cgctcgcgcg
      301 cgtaacatgc ataaagctgc aaaaatgatc tgcggagact ttaaaggcca gtttcctgac
      361 aatctggagg ctatgcaaag cttgtcaggc atcgggcgct cgaccgccgc cgctatcttg
      421 agtattgcct cgaatcagcc gcaggctatt ctgaacggaa atgtgaagcg cgtcttgact
      481 cgtgtgttcg tggtggatgg ttggcctgga aaggccgatg tccttaaaaa gttgtggtta
      541 ttggctgagc agacaacacc tgcaagccgc aacgctgatt atacacaagc aattatggac
      601 cttggagcaa ccttatgtac tcgttctaag ccgcgttgtg aagactgtcc tctaaaagcc
      661 gactgtttag cgttccaaca ggggaatcag acagtctacc ctacttccaa accaaagaaa
      721 gttatgcccg aaaaacaggc catcatggta attctaaaga attctaaaca ggaagtcttc
      781 atgcaaaaac gcccgccagt gggcatctgg ggatctttgt ggtgcttccc ccagtttgaa
      841 aagcgcgcct ttgctgagga gtggttgaaa tctaattatg gtaagttaat tcaggaaagt
      901 attgcgcttg cagaaatcaa acacactttt tcgcattttc atctgtatat ccagccatta
      961 atcattaatg ttgaaacacc aattgagttg ggtgtcatgg aggacaacga atctctttgg
     1021 tataacatta cgacggagtt taatggtggt cttgcggctc ccgtccaaac tctaattaac
     1081 attaccaaga acatcgagaa agagattgag aatggaacca acggctgact gcagccaagc
     1141 ttggctgttt tggcggatga gagaagattt tcagcctgat acagattaaa tcagaacgca
     1201 gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcggtggtc ccacctgacc
     1261 ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg tctccccatg
     1321 cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
     1381 tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga
     1441 gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa
     1501 actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta
     1561 caaactcttt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     1621 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     1681 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     1741 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     1801 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     1861 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga
     1921 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     1981 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     2041 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     2101 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     2161 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     2221 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     2281 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     2341 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     2401 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     2461 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     2521 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     2581 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     2641 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     2701 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     2761 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     2821 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     2881 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     2941 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     3001 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     3061 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     3121 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     3181 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     3241 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     3301 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     3361 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     3421 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     3481 tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg
     3541 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     3601 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     3661 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     3721 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     3781 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     3841 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     3901 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     3961 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     4021 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     4081 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     4141 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     4201 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     4261 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     4321 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg
     4381 ctgcccgaga tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc
     4441 caagggttgg tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga
     4501 gtggtgaatc cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc
     4561 atgcaccgcg acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg
     4621 ccaacccgtt ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccaa
     4681 tgatcgaagt taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt
     4741 catctacctg cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga
     4801 gaagaatcat aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc
     4861 ccagcgcgtc ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg
     4921 cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca
     4981 ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg
     5041 ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag
     5101 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     5161 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     5221 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     5281 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     5341 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     5401 cggtgatgcc ggccacgatg cgtccggcgt agaggatccg ggcttatcga ctgcacggtg
     5461 caccaatgct tctggcgtca ggcagccatc ggaagctgtg gtatggctgt gcaggtcgta
     5521 aatcactgca taattcgtgt cgctcaaggc gcactcccgt tctggataat gttttttgcg
     5581 ccgacatcat aacggttctg gcaaatattc tgaaatgagc tgttgacaat taatcatcgg
     5641 ctcgtataat gtgtggaatt gtgagcggat aacaatttca cacaggaaa
//
