LOCUS       sequence-203152-        4979 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Small IncP-based broad host range replicating vector..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4979)
  AUTHORS   Puri AW, Owen S, Chu F, Chavkin T, Beck DA, Kalyuzhnaya MG, Lidstrom
            ME
  TITLE     Genetic tools for the industrially promising methanotroph 
            Methylomicrobium buryatense.
  JOURNAL   Appl Environ Microbiol. 2014 Dec 29. pii: AEM.03795-14.
  PUBMED    25548049
REFERENCE   2  (bases 1 to 4979)
  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: "Appl 
            Environ Microbiol. 2014 Dec 29. pii: AEM.03795-14."
COMMENT     Sequence Label: pAWP78
FEATURES             Location/Qualifiers
     source          1..4979
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     oriT            1..110
                     /note="incP origin of transfer"
     rep_origin      263..894
                     /label=oriV
                     /note="incP origin of replication"
     rep_origin      1203..1791
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1692..1711
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2037..2852)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2037..2852)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2760..2779
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2844..2863
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             complement(3156..4304)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     CDS             complement(4576..4947)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSL*AYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
ORIGIN
        1 ccggccagcc tcgcagagca ggattcccgt tgagcaccgc caggtgcgaa taagggacag
       61 tgaagaagga acacccgctc gcgggtgggc ctacttcacc tatcctgccc ggctgacgcc
      121 gttggataca ccaaggaaag tctacacgaa ccctttggca aaatcctgta tatcgtgcga
      181 aaaaggatgg atataccgaa aaaatcgcta taatgacccc gaagcagggt tatgcagcgg
      241 aaaagatccg tcgacccttt ccgacgctca ccgggctggt tgccctcgcc gctgggctgg
      301 cggccgtcta tggccctgca aacgcgccag aaacgccgtc gaagccgtgt gcgagacacc
      361 gcggccgccg gcgttgtgga tacctcgcgg aaaacttggc cctcactgac agatgagggg
      421 cggacgttga cacttgaggg gccgactcac ccggcgcggc gttgacagat gaggggcagg
      481 ctcgatttcg gccggcgacg tggagctggc cagcctcgca aatcggcgaa aacgcctgat
      541 tttacgcgag tttcccacag atgatgtgga caagcctggg gataagtgcc ctgcggtatt
      601 gacacttgag gggcgcgact actgacagat gaggggcgcg atccttgaca cttgaggggc
      661 agagtgctga cagatgaggg gcgcacctat tgacatttga ggggctgtcc acaggcagaa
      721 aatccagcat ttgcaagggt ttccgcccgt ttttcggcca ccgctaacct gtcttttaac
      781 ctgcttttaa accaatattt ataaaccttg tttttaacca gggctgcgcc ctgtgcgcgt
      841 gaccgcgcac gccgaagggg ggtgcccccc cttctcgaac cctcccggcc cgctaacgcg
      901 ggcctcccat ccccccaggg gctgcgcccc tcggccgcga acggcctcac cccaaaaatg
      961 gcagccaagc tgacccgcta gggacgtgaa gtcgattcct tcgatggtta gcaatcaaag
     1021 actcagagtg ctgtctggag cgtgaatcta acggtacgta tctcgattgc tcggtcgcta
     1081 ttcgcactct gcgaaagttc gtaccgctca ttcactaggt tgcgaatcat gaccaaaatc
     1141 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     1201 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     1261 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     1321 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     1381 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     1441 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     1501 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     1561 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     1621 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     1681 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     1741 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     1801 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     1861 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgttgtcggg
     1921 aagatgcgtg atctgatcct tcaactcagc aaaagttcga tttattcaac aaagccgccg
     1981 tcccgtcaag tcagcgtaat gctctgccag tgttacaacc aattaaccaa ttctgattag
     2041 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca
     2101 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
     2161 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt
     2221 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa
     2281 tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac aggccagcca
     2341 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc
     2401 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc
     2461 aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct
     2521 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca
     2581 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt
     2641 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac
     2701 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta
     2761 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggcctc
     2821 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa
     2881 gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga
     2941 ttttgagaca caacgtggct ttcgacttcc ggcaagctat acgcgcccta gaattgtcaa
     3001 ttttaatcct ctgtttatcg gcagttcgta gagcgcgccg tgcgtcccga gcgatactga
     3061 gcgaagcaag tgcgtcgagc agtgcccgct tgttcctgaa atgccagtaa agcgctggct
     3121 gctgaacccc cagccggaac tgaccccaca aggccctagc gtttgcaatg caccaggtca
     3181 tcattgaccc aggcgtgttc caccaggccg ctgcctcgca actcttcgca ggcttcgccg
     3241 acctgctcgc gccacttctt cacgcgggtg gaatccgatc cgcacatgag gcggaaggtt
     3301 tccagcttga gcgggtacgg ctcccggtgc gagctgaaat agtcgaacat ccgtcgggcc
     3361 gtcggcgaca gcttgcggta cttctcccat atgaatttcg tgtagtggtc gccagcaaac
     3421 agcacgacga tttcctcgtc gatcaggacc tggcaacggg acgttttctt gccacggtcc
     3481 aggacgcgga agcggtgcag cagcgacacc gattccaggt gcccaacgcg gtcggacgtg
     3541 aagcccatcg ccgtcgcctg taggcgcgac aggcattcct cggccttcgt gtaataccgg
     3601 ccattgatcg accagcccag gtcctggcaa agctcgtaga acgtgaaggt gatcggctcg
     3661 ccgatagggg tgcgcttcgc gtactccaac acctgctgcc acaccagttc gtcatcgtcg
     3721 gcccgcagct cgacgccggt gtaggtgatc ttcacgtcct tgttgacgtg gaaaatgacc
     3781 ttgttttgca gcgcctcgcg cgggattttc ttgttgcgcg tggtgaacag ggcagagcgg
     3841 gccgtgtcgt ttggcatcgc tcgcatcgtg tccggccacg gcgcaatatc gaacaaggaa
     3901 agctgcattt ccttgatctg ctgcttcgtg tgtttcagca acgcggcctg cttggcctcg
     3961 ctgacctgtt ttgccaggtc ctcgccggcg gtttttcgct tcttggtcgt catagttcct
     4021 cgcgtgtcga tggtcatcga cttcgccaaa cctgccgcct cctgttcgag acgacgcgaa
     4081 cgctccacgg cggccgatgg cgcgggcagg gcagggggag ccagttgcac gctgtcgcgc
     4141 tcgatcttgg ccgtagcttg ctggaccatc gagccgacgg actggaaggt ttcgcggggc
     4201 gcacgcatga cggtgcggct tgcgatggtt tcggcatcct cggcggaaaa ccccgcgtcg
     4261 atcagttctt gcctgtatgc cttccggtca aacgtccgat tcattcaccc tccttgcggg
     4321 attgccccga ctcacgccgg ggcaatgtgc ccttattcct gatttgaccc gcctggtgcc
     4381 ttggtgtcca gataatccac cttatcggca atgaagtcgg tcccgtagac cgtctggccg
     4441 tccttctcgt acttggtatt ccgaatcttg ccctgcacga ataccagctc cgcgaagtcg
     4501 ctcttcttga tggagcgcat ggggacgtgc ttggcaatca cgcgcacccc ccggccgttt
     4561 tagcggctaa aaaagtcatg gctctgccct cgggcggacc acgcccatca tgaccttgcc
     4621 aagctcgtcc tgcttctctt cgatcttcgc cagcagggcg aggatcgtgg catcaccgaa
     4681 ccgcgccgtg cgcgggtcgt cggtgagcca gagtttcagc aggccgccca ggcggcccag
     4741 gtcgccattg atgcgggcca gctcgcggac gtgctcatag tccacgacgc ccgtgatttt
     4801 gtagccctgg ccgacggcca gcaggtaggc ctacaggctc atgccggccg ccgccgcctt
     4861 ttcctcaatc gctcttcgtt cgtctggaag gcagtacacc ttgataggtg ggctgccctt
     4921 cctggttggc ttggtttcat cagccatccg cttgccctca tctgttacgc cggcggtag
//
