LOCUS       sequence-420128-        5635 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pkk223 with Rhodobacteraceae MutY containing D146N and E45Q 
            mutations to reduce catalytic activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5635)
  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 5635)
  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_RbMutY_D146N_E45Q
FEATURES             Location/Qualifiers
     source          1..5635
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1126..1143)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1296..1382
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1474..1501
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1520..1611
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1612..1680,1681..2472)
                     /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(1830..1849)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2643..3231
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3132..3151
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3385..3402
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3417..3557
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3483..3502)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3643..3665
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3659..3850)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5308..5327
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(5574..5579,5580..5595,5596..5602)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    5610..5626
                     /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     join(5615..5635,1..2)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 ggaaacagaa ttcggtgacc atgcgtgacg aagatctttc tcgcgcactg cttgattggt
       61 acgacggcca tgcaagagcg atgccgtggc gtgttggacc cagcgatcgt cgtgggggga
      121 tccgtcccga cgcttacgct gtatggctta gtcaagtaat gttgcagcaa acgacagttg
      181 ctgctgtgcg cgactacttc cgccgtttta ctacgcgttg gcctaccgta gccgatcttg
      241 cagcggctga agatgctgat gtaatggggg agtgggcggg actggggtat tacgcacgcg
      301 cgcgtaactt actgaaatgc gcacgtgttg ttgtggctga acacggaggg cacttcccgg
      361 atactcgcga cggattactg aaattaccag ggattggacc ttacacagcg gccgcgattg
      421 cagctattgc atttgatgag cccgcggtcg tagtcaacgg aaatgttgag cgcgtaatgg
      481 cgcgtctgtt tcaaattgaa acgccattgc ctggtgcaaa gcctgaatta acggaactgg
      541 ctggccgctt gactccggat attcgtccgg gtgaccacgc ccaagctgtc atggacctgg
      601 gggcgacgat ttgcacgccc aaatcaccgg cgtgcgggat ctgtcccgta atgacgggtt
      661 gtacggctag agccgcaggg gcgcaagcag acttacctcg caaaacaccc aagaagccta
      721 aaccgactcg tcatgggatc gcttatctag gccgccgtgc cgatggagcg tggcttttag
      781 aacgtcgccc cgataagggg ttgttgggcg gcatgcttgg ctggccagga ggtgattggg
      841 ctgaaactgc aacggaggca ccccccattg acgctccctg gcgtgacccg ggggcggaag
      901 tgcgccatac gtttacccat ttccatttgc gccttgcctt gcgtgtggcg gacttaccag
      961 cgtctgctac gccacgcaca gggcagttca tcccacatca tgcgttccgc cctggacagc
     1021 tacccactgt gatgcgtaaa gcctacgact tggcaagtgc aaccttttta gggaattgac
     1081 tgcagccaag cttggctgtt ttggcggatg agagaagatt ttcagcctga tacagattaa
     1141 atcagaacgc agaagcggtc tgataaaaca gaatttgcct ggcggcagta gcgcggtggt
     1201 cccacctgac cccatgccga actcagaagt gaaacgccgt agcgccgatg gtagtgtggg
     1261 gtctccccat gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga
     1321 aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa
     1381 atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac
     1441 gcccgccata aactgccagg catcaaatta agcagaaggc catcctgacg gatggccttt
     1501 ttgcgtttct acaaactctt ttgtttattt ttctaaatac attcaaatat gtatccgctc
     1561 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     1621 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     1681 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     1741 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     1801 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac
     1861 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     1921 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     1981 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     2041 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     2101 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     2161 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     2221 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     2281 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     2341 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     2401 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     2461 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     2521 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     2581 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     2641 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     2701 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     2761 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     2821 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     2881 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     2941 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     3001 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     3061 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     3121 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     3181 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     3241 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     3301 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     3361 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     3421 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc
     3481 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg
     3541 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     3601 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     3661 agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt
     3721 ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct
     3781 ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct
     3841 gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga
     3901 tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg
     3961 aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc
     4021 agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc
     4081 atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac
     4141 tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc
     4201 agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc
     4261 aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtggcc
     4321 aggacccaac gctgcccgag atgcgccgcg tgcggctgct ggagatggcg gacgcgatgg
     4381 atatgttctg ccaagggttg gtttgcgcat tcacagttct ccgcaagaat tgattggctc
     4441 caattcttgg agtggtgaat ccgttagcga ggtgccgccg gcttccattc aggtcgaggt
     4501 ggcccggctc catgcaccgc gacgcaacgc ggggaggcag acaaggtata gggcggcgcc
     4561 tacaatccat gccaacccgt tccatgtgct cgccgaggcg gcataaatcg ccgtgacgat
     4621 cagcggtcca atgatcgaag ttaggctggt aagagccgcg agcgatcctt gaagctgtcc
     4681 ctgatggtcg tcatctacct gcctggacag catggcctgc aacgcgggca tcccgatgcc
     4741 gccggaagcg agaagaatca taatggggaa ggccatccag cctcgcgtcg cgaacgccag
     4801 caagacgtag cccagcgcgt cggccgccat gccggcgata atggcctgct tctcgccgaa
     4861 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
     4921 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
     4981 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
     5041 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     5101 gggcatcggt cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     5161 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
     5221 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
     5281 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
     5341 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcc gggcttatcg
     5401 actgcacggt gcaccaatgc ttctggcgtc aggcagccat cggaagctgt ggtatggctg
     5461 tgcaggtcgt aaatcactgc ataattcgtg tcgctcaagg cgcactcccg ttctggataa
     5521 tgttttttgc gccgacatca taacggttct ggcaaatatt ctgaaatgag ctgttgacaa
     5581 ttaatcatcg gctcgtataa tgtgtggaat tgtgagcgga taacaatttc acaca
//
