LOCUS       sequence-429379-        5629 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pkk223 with E coli MutY insert.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5629)
  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 5629)
  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_EcMutY
FEATURES             Location/Qualifiers
     source          1..5629
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1120..1137)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1290..1376
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1468..1495
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1514..1605
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1606..1674,1675..2466)
                     /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(1824..1843)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2637..3225
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3126..3145
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3379..3396
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3411..3551
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3477..3496)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3637..3659
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3653..3844)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5302..5321
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(5568..5573,5574..5589,5590..5596)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    5604..5620
                     /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(5609..5629,1..2)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 ggaaacagaa ttcggtgacc atgcaagcgt cgcaattttc agcccaggtt ctggactggt
       61 acgataaata cgggcgaaaa actctgccct ggcaaattga caagacgccc tacaaagtat
      121 ggctctcaga agtgatgttg caacaaactc aggttgcgac cgttatcccc tattttgaac
      181 gctttatggc gcgcttcccg acggtgaccg atctcgccaa tgcgccgctc gacgaagttc
      241 tccacttgtg gaccgggctt ggctattacg cccgcgcgcg caatctgcat aaagcggcac
      301 aacaagtggc gaccttacac ggcggtaaat tcccggaaac ctttgaggaa gttgcagcac
      361 tgccgggcgt cgggcgttcc accgcaggcg cgattctctc gctttctctg ggtaagcact
      421 ttccgattct cgacggtaac gtcaaacgcg tgctggcgcg ctgctatgct gtaagcggct
      481 ggcctgggaa aaaagaggtc gagaataaat tatggagttt gagcgagcag gtgacgcccg
      541 cggttggcgt ggaacggttt aatcaggcga tgatggattt gggtgcgatg atttgtacgc
      601 gctcgaaacc gaaatgttcg ctctgtccgc tacaaaacgg atgtattgcc gccgccaaca
      661 atagctgggc gctttatccg ggcaaaaaac cgaaacagac gctgccggag cgcaccggct
      721 actttttgct attacagcac gaagatgaag tattgctggc gcagcgtccg ccgagcggat
      781 tgtggggcgg tttatactgt ttcccgcagt ttgccgacga agaaagtttg cggcagtggc
      841 tggcgcaacg gcagattgct gccgataacc tgacgcaact gaccgcgttt cggcatacct
      901 tcagccattt ccacttagat attgtgccta tgtggcttcc cgtgtcgtca ttcaccggct
      961 gcatggatga aggcaatgcg ctctggtata acttagcgca accgccgtca gttggcctag
     1021 cggctcccgt ggagcgtttg ttacagcagt tacgcactgg cgcgccggtt tagctgcagc
     1081 caagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga ttaaatcaga
     1141 acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg tggtcccacc
     1201 tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg tggggtctcc
     1261 ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag tcgaaagact
     1321 gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc
     1381 cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc
     1441 cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc ctttttgcgt
     1501 ttctacaaac tcttttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     1561 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     1621 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     1681 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     1741 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     1801 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg
     1861 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     1921 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     1981 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     2041 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     2101 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     2161 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     2221 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     2281 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     2341 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     2401 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     2461 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     2521 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     2581 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     2641 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     2701 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     2761 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     2821 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     2881 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     2941 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgaactac
     3001 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     3061 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     3121 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     3181 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     3241 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     3301 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     3361 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     3421 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca
     3481 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     3541 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     3601 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     3661 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
     3721 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
     3781 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
     3841 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac
     3901 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt
     3961 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc
     4021 aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg
     4081 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg
     4141 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc
     4201 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc
     4261 gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggccaggacc
     4321 caacgctgcc cgagatgcgc cgcgtgcggc tgctggagat ggcggacgcg atggatatgt
     4381 tctgccaagg gttggtttgc gcattcacag ttctccgcaa gaattgattg gctccaattc
     4441 ttggagtggt gaatccgtta gcgaggtgcc gccggcttcc attcaggtcg aggtggcccg
     4501 gctccatgca ccgcgacgca acgcggggag gcagacaagg tatagggcgg cgcctacaat
     4561 ccatgccaac ccgttccatg tgctcgccga ggcggcataa atcgccgtga cgatcagcgg
     4621 tccaatgatc gaagttaggc tggtaagagc cgcgagcgat ccttgaagct gtccctgatg
     4681 gtcgtcatct acctgcctgg acagcatggc ctgcaacgcg ggcatcccga tgccgccgga
     4741 agcgagaaga atcataatgg ggaaggccat ccagcctcgc gtcgcgaacg ccagcaagac
     4801 gtagcccagc gcgtcggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt
     4861 ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag
     4921 cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag
     4981 cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac
     5041 gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     5101 cggtcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     5161 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     5221 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     5281 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     5341 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atccgggctt atcgactgca
     5401 cggtgcacca atgcttctgg cgtcaggcag ccatcggaag ctgtggtatg gctgtgcagg
     5461 tcgtaaatca ctgcataatt cgtgtcgctc aaggcgcact cccgttctgg ataatgtttt
     5521 ttgcgccgac atcataacgg ttctggcaaa tattctgaaa tgagctgttg acaattaatc
     5581 atcggctcgt ataatgtgtg gaattgtgag cggataacaa tttcacaca
//
