LOCUS       sequence-99456-e        5756 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  IncP-based replicating vector expressing dTomato under tac 
            promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5756)
  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 5756)
  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: pAWP89
FEATURES             Location/Qualifiers
     source          1..5756
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      11..642
                     /label=oriV
                     /note="incP origin of replication"
     rep_origin      951..1539
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1440..1459
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(1676..1681,1682..1697,1698..1704)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     CDS             1733..2434
                     /codon_start=1
                     /product="dimeric variant of DsRed fluorescent protein 
                     (Shaner et al., 2004)"
                     /label=dTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLYGMDELYK"
     primer_bind     complement(1866..1884)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2134..2153
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2328..2351
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             complement(2562..3377)
                     /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(2562..3377)
                     /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     3285..3304
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3369..3388
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             complement(3681..4829)
                     /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(5101..5472)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSL*AYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            5505..5614
                     /note="incP origin of transfer"
ORIGIN
        1 gaccctttcc gacgctcacc gggctggttg ccctcgccgc tgggctggcg gccgtctatg
       61 gccctgcaaa cgcgccagaa acgccgtcga agccgtgtgc gagacaccgc ggccgccggc
      121 gttgtggata cctcgcggaa aacttggccc tcactgacag atgaggggcg gacgttgaca
      181 cttgaggggc cgactcaccc ggcgcggcgt tgacagatga ggggcaggct cgatttcggc
      241 cggcgacgtg gagctggcca gcctcgcaaa tcggcgaaaa cgcctgattt tacgcgagtt
      301 tcccacagat gatgtggaca agcctgggga taagtgccct gcggtattga cacttgaggg
      361 gcgcgactac tgacagatga ggggcgcgat ccttgacact tgaggggcag agtgctgaca
      421 gatgaggggc gcacctattg acatttgagg ggctgtccac aggcagaaaa tccagcattt
      481 gcaagggttt ccgcccgttt ttcggccacc gctaacctgt cttttaacct gcttttaaac
      541 caatatttat aaaccttgtt tttaaccagg gctgcgccct gtgcgcgtga ccgcgcacgc
      601 cgaagggggg tgccccccct tctcgaaccc tcccggcccg ctaacgcggg cctcccatcc
      661 ccccaggggc tgcgcccctc ggccgcgaac ggcctcaccc caaaaatggc agccaagctg
      721 acccgctagg gacgtgaagt cgattccttc gatggttagc aatcaaagac tcagagtgct
      781 gtctggagcg tgaatctaac ggtacgtatc tcgattgctc ggtcgctatt cgcactctgc
      841 gaaagttcgt accgctcatt cactaggttg cgaatcatga ccaaaatccc ttaacgtgag
      901 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
      961 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     1021 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     1081 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     1141 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     1201 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     1261 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     1321 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     1381 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     1441 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     1501 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     1561 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     1621 gattctgtgg ataaccgtat taccgccttt gagtgagctg ctctgaaatg agctgttgac
     1681 aattaatcat cggctcgtat aatgtgtgga ggtattcaca caggaaacag ctatggtgag
     1741 caagggcgag gaggtcatca aagagttcat gcgcttcaag gtgcgcatgg agggctccat
     1801 gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca
     1861 gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg acatcctgtc
     1921 cccccagttc atgtacggct ccaaggcgta cgtgaagcac cccgccgaca tccccgatta
     1981 caagaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg
     2041 cggtctggtg accgtgaccc aggactcctc cctgcaggac ggcacgctga tctacaaggt
     2101 gaagatgcgc ggcaccaact tcccccccga cggccccgta atgcagaaga agaccatggg
     2161 ctgggaggcc tccaccgagc gcctgtaccc ccgcgacggc gtgctgaagg gcgagatcca
     2221 ccaggccctg aagctgaagg acggcggcca ctacctggtg gagttcaaga ccatctacat
     2281 ggccaagaag cccgtgcaac tgcccggcta ctactacgtg gacaccaagc tggacatcac
     2341 ctcccacaac gaggactaca ccatcgtgga acagtacgag cgctccgagg gccgccacca
     2401 cctgttcctg tacggcatgg acgagctgta caagtagttg tcgggaagat gcgtgatctg
     2461 atccttccac tcagcaaaag ttcgatttat tcaacaaagc cgccgtcccg tcaagtcagc
     2521 gtaatgctct gccagtgtta caaccaatta accaattctg attagaaaaa ctcatcgagc
     2581 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
     2641 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
     2701 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
     2761 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
     2821 aaaagcttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     2881 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
     2941 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     3001 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     3061 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     3121 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     3181 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     3241 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     3301 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctcgagca agacgtttcc
     3361 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
     3421 gttcatgatg atatattttt atcttgtgca atgtaacatc agagattttg agacacaacg
     3481 tggctttcga cttccggcaa gctatacgcg ccctagaatt gtcaatttta atcctctgtt
     3541 tatcggcagt tcgtagagcg cgccgtgcgt cccgagcgat actgagcgaa gcaagtgcgt
     3601 cgagcagtgc ccgcttgttc ctgaaatgcc agtaaagcgc tggctgctga acccccagcc
     3661 ggaactgacc ccacaaggcc ctagcgtttg caatgcacca ggtcatcatt gacccaggcg
     3721 tgttccacca ggccgctgcc tcgcaactct tcgcaggctt cgccgacctg ctcgcgccac
     3781 ttcttcacgc gggtggaatc cgatccgcac atgaggcgga aggtttccag cttgagcggg
     3841 tacggctccc ggtgcgagct gaaatagtcg aacatccgtc gggccgtcgg cgacagcttg
     3901 cggtacttct cccatatgaa tttcgtgtag tggtcgccag caaacagcac gacgatttcc
     3961 tcgtcgatca ggacctggca acgggacgtt ttcttgccac ggtccaggac gcggaagcgg
     4021 tgcagcagcg acaccgattc caggtgccca acgcggtcgg acgtgaagcc catcgccgtc
     4081 gcctgtaggc gcgacaggca ttcctcggcc ttcgtgtaat accggccatt gatcgaccag
     4141 cccaggtcct ggcaaagctc gtagaacgtg aaggtgatcg gctcgccgat aggggtgcgc
     4201 ttcgcgtact ccaacacctg ctgccacacc agttcgtcat cgtcggcccg cagctcgacg
     4261 ccggtgtagg tgatcttcac gtccttgttg acgtggaaaa tgaccttgtt ttgcagcgcc
     4321 tcgcgcggga ttttcttgtt gcgcgtggtg aacagggcag agcgggccgt gtcgtttggc
     4381 atcgctcgca tcgtgtccgg ccacggcgca atatcgaaca aggaaagctg catttccttg
     4441 atctgctgct tcgtgtgttt cagcaacgcg gcctgcttgg cctcgctgac ctgttttgcc
     4501 aggtcctcgc cggcggtttt tcgcttcttg gtcgtcatag ttcctcgcgt gtcgatggtc
     4561 atcgacttcg ccaaacctgc cgcctcctgt tcgagacgac gcgaacgctc cacggcggcc
     4621 gatggcgcgg gcagggcagg gggagccagt tgcacgctgt cgcgctcgat cttggccgta
     4681 gcttgctgga ccatcgagcc gacggactgg aaggtttcgc ggggcgcacg catgacggtg
     4741 cggcttgcga tggtttcggc atcctcggcg gaaaaccccg cgtcgatcag ttcttgcctg
     4801 tatgccttcc ggtcaaacgt ccgattcatt caccctcctt gcgggattgc cccgactcac
     4861 gccggggcaa tgtgccctta ttcctgattt gacccgcctg gtgccttggt gtccagataa
     4921 tccaccttat cggcaatgaa gtcggtcccg tagaccgtct ggccgtcctt ctcgtacttg
     4981 gtattccgaa tcttgccctg cacgaatacc agctccgcga agtcgctctt cttgatggag
     5041 cgcatgggga cgtgcttggc aatcacgcgc accccccggc cgttttagcg gctaaaaaag
     5101 tcatggctct gccctcgggc ggaccacgcc catcatgacc ttgccaagct cgtcctgctt
     5161 ctcttcgatc ttcgccagca gggcgaggat cgtggcatca ccgaaccgcg ccgtgcgcgg
     5221 gtcgtcggtg agccagagtt tcagcaggcc gcccaggcgg cccaggtcgc cattgatgcg
     5281 ggccagctcg cggacgtgct catagtccac gacgcccgtg attttgtagc cctggccgac
     5341 ggccagcagg taggcctaca ggctcatgcc ggccgccgcc gccttttcct caatcgctct
     5401 tcgttcgtct ggaaggcagt acaccttgat aggtgggctg cccttcctgg ttggcttggt
     5461 ttcatcagcc atccgcttgc cctcatctgt tacgccggcg gtagccggcc agcctcgcag
     5521 agcaggattc ccgttgagca ccgccaggtg cgaataaggg acagtgaaga aggaacaccc
     5581 gctcgcgggt gggcctactt cacctatcct gcccggctga cgccgttgga tacaccaagg
     5641 aaagtctaca cgaacccttt ggcaaaatcc tgtatatcgt gcgaaaaagg atggatatac
     5701 cgaaaaaatc gctataatga ccccgaagca gggttatgca gcggaaaaga tccgtc
//
