LOCUS       sequence-420129-        5635 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pkk223 with Rhodobacteraceae MutY containing F298A, S299A, and H300A
            mutations to reduce recognition of OG:A.
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_F298A_T299A_H300A
FEATURES             Location/Qualifiers
     source          1..5635
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1117..1134)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1287..1373
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1465..1492
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1511..1602
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1603..1671,1672..2463)
                     /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(1821..1840)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2634..3222
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3123..3142
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3376..3393
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3408..3548
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3474..3493)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3634..3656
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3650..3841)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5299..5318
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(5565..5570,5571..5586,5587..5593)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    5601..5617
                     /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     5606..5628
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 attcggtgac catgcgtgac gaagatcttt ctcgcgcact gcttgattgg tacgacggcc
       61 atgcaagagc gatgccgtgg cgtgttggac ccagcgatcg tcgtgggggg atccgtcccg
      121 acgcttacgc tgtatggctt agtgaagtaa tgttgcagca aacgacagtt gctgctgtgc
      181 gcgactactt ccgccgtttt actacgcgtt ggcctaccgt agccgatctt gcagcggctg
      241 aagatgctga tgtaatgggg gagtgggcgg gactggggta ttacgcacgc gcgcgtaact
      301 tactgaaatg cgcacgtgtt gttgtggctg aacacggagg gcacttcccg gatactcgcg
      361 acggattact gaaattacca gggattggac cttacacagc ggccgcgatt gcagctattg
      421 catttgatga gcccgcggtc gtagtcgacg gaaatgttga gcgcgtaatg gcgcgtctgt
      481 ttcaaattga aacgccattg cctggtgcaa agcctgaatt aacggaactg gctggccgct
      541 tgactccgga tattcgtccg ggtgaccacg cccaagctgt catggacctg ggggcgacga
      601 tttgcacgcc caaatcaccg gcgtgcggga tctgtcccgt aatgacgggt tgtacggcta
      661 gagccgcagg ggcgcaagca gacttacctc gcaaaacacc caagaagcct aaaccgactc
      721 gtcatgggat cgcttatcta ggccgccgtg ccgatggagc gtggctttta gaacgtcgcc
      781 ccgataaggg gttgttgggc ggcatgcttg gctggccagg aggtgattgg gctgaaactg
      841 caacggaggc accccccatt gacgctccct ggcgtgaccc gggggcggaa gtgcgccata
      901 cggctgccgc tttccatttg cgccttgcct tgcgtgtggc ggacttacca gcgtctgcta
      961 cgccacgcac agggcagttc atcccacatc atgcgttccg ccctggacag ctacccactg
     1021 tgatgcgtaa agcctacgac ttggcaagtg caaccttttt agggaattga ctgcagccaa
     1081 gcttggctgt tttggcggat gagagaagat tttcagcctg atacagatta aatcagaacg
     1141 cagaagcggt ctgataaaac agaatttgcc tggcggcagt agcgcggtgg tcccacctga
     1201 ccccatgccg aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca
     1261 tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg
     1321 cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgg
     1381 gagcggattt gaacgttgcg aagcaacggc ccggagggtg gcgggcagga cgcccgccat
     1441 aaactgccag gcatcaaatt aagcagaagg ccatcctgac ggatggcctt tttgcgtttc
     1501 tacaaactct tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     1561 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     1621 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     1681 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     1741 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     1801 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa
     1861 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     1921 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     1981 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     2041 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     2101 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     2161 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     2221 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     2281 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     2341 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     2401 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     2461 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     2521 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     2581 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     2641 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     2701 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     2761 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     2821 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     2881 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     2941 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     3001 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     3061 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     3121 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     3181 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     3241 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     3301 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     3361 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     3421 tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc
     3481 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     3541 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     3601 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     3661 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa
     3721 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
     3781 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
     3841 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
     3901 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg
     3961 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat
     4021 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga
     4081 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa
     4141 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt
     4201 cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc
     4261 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggc caggacccaa
     4321 cgctgcccga gatgcgccgc gtgcggctgc tggagatggc ggacgcgatg gatatgttct
     4381 gccaagggtt ggtttgcgca ttcacagttc tccgcaagaa ttgattggct ccaattcttg
     4441 gagtggtgaa tccgttagcg aggtgccgcc ggcttccatt caggtcgagg tggcccggct
     4501 ccatgcaccg cgacgcaacg cggggaggca gacaaggtat agggcggcgc ctacaatcca
     4561 tgccaacccg ttccatgtgc tcgccgaggc ggcataaatc gccgtgacga tcagcggtcc
     4621 aatgatcgaa gttaggctgg taagagccgc gagcgatcct tgaagctgtc cctgatggtc
     4681 gtcatctacc tgcctggaca gcatggcctg caacgcgggc atcccgatgc cgccggaagc
     4741 gagaagaatc ataatgggga aggccatcca gcctcgcgtc gcgaacgcca gcaagacgta
     4801 gcccagcgcg tcggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     4861 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     4921 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     4981 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     5041 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     5101 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
     5161 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
     5221 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
     5281 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
     5341 gccggtgatg ccggccacga tgcgtccggc gtagaggatc cgggcttatc gactgcacgg
     5401 tgcaccaatg cttctggcgt caggcagcca tcggaagctg tggtatggct gtgcaggtcg
     5461 taaatcactg cataattcgt gtcgctcaag gcgcactccc gttctggata atgttttttg
     5521 cgccgacatc ataacggttc tggcaaatat tctgaaatga gctgttgaca attaatcatc
     5581 ggctcgtata atgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acaga
//
