LOCUS       sequence-429380-        4585 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pkk223 backbone plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4585)
  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 4585)
  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_Null
FEATURES             Location/Qualifiers
     source          1..4585
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(76..93)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      246..332
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      424..451
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        470..561
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(562..630,631..1422)
                     /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(780..799)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1593..2181
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2082..2101
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2335..2352
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2367..2507
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2433..2452)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2593..2615
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2609..2800)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     4258..4277
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(4524..4529,4530..4545,4546..4552)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    4560..4576
                     /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(4565..4585,1..2)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 ggaaacagaa ttcccgggga tccgtcgacc tgcagccaag cttggctgtt ttggcggatg
       61 agagaagatt ttcagcctga tacagattaa atcagaacgc agaagcggtc tgataaaaca
      121 gaatttgcct ggcggcagta gcgcggtggt cccacctgac cccatgccga actcagaagt
      181 gaaacgccgt agcgccgatg gtagtgtggg gtctccccat gcgagagtag ggaactgcca
      241 ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt
      301 tgtcggtgaa cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga
      361 agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaatta
      421 agcagaaggc catcctgacg gatggccttt ttgcgtttct acaaactctt ttgtttattt
      481 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
      541 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
      601 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
      661 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
      721 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
      781 ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata
      841 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
      901 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
      961 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     1021 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     1081 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     1141 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     1201 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     1261 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     1321 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     1381 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     1441 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     1501 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     1561 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     1621 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     1681 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     1741 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     1801 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     1861 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     1921 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     1981 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     2041 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     2101 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     2161 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     2221 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     2281 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     2341 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     2401 ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     2461 agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc
     2521 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     2581 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     2641 cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct
     2701 gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa
     2761 agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg
     2821 gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg
     2881 ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat
     2941 ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag
     3001 atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg
     3061 tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc
     3121 atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta
     3181 tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg
     3241 acaggagcac gatcatgcgc acccgtggcc aggacccaac gctgcccgag atgcgccgcg
     3301 tgcggctgct ggagatggcg gacgcgatgg atatgttctg ccaagggttg gtttgcgcat
     3361 tcacagttct ccgcaagaat tgattggctc caattcttgg agtggtgaat ccgttagcga
     3421 ggtgccgccg gcttccattc aggtcgaggt ggcccggctc catgcaccgc gacgcaacgc
     3481 ggggaggcag acaaggtata gggcggcgcc tacaatccat gccaacccgt tccatgtgct
     3541 cgccgaggcg gcataaatcg ccgtgacgat cagcggtcca atgatcgaag ttaggctggt
     3601 aagagccgcg agcgatcctt gaagctgtcc ctgatggtcg tcatctacct gcctggacag
     3661 catggcctgc aacgcgggca tcccgatgcc gccggaagcg agaagaatca taatggggaa
     3721 ggccatccag cctcgcgtcg cgaacgccag caagacgtag cccagcgcgt cggccgccat
     3781 gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc
     3841 ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct
     3901 ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag
     3961 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
     4021 gaaggagctg actgggttga aggctctcaa gggcatcggt cgacgctctc ccttatgcga
     4081 ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag
     4141 gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat
     4201 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt
     4261 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat
     4321 gcgtccggcg tagaggatcc gggcttatcg actgcacggt gcaccaatgc ttctggcgtc
     4381 aggcagccat cggaagctgt ggtatggctg tgcaggtcgt aaatcactgc ataattcgtg
     4441 tcgctcaagg cgcactcccg ttctggataa tgttttttgc gccgacatca taacggttct
     4501 ggcaaatatt ctgaaatgag ctgttgacaa ttaatcatcg gctcgtataa tgtgtggaat
     4561 tgtgagcgga taacaatttc acaca
//
