LOCUS       sequence-249045-        7627 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  IncP-based broad host range vector used for inducible expression of 
            genes in methanotrophic bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7627)
  AUTHORS   Henard CA, Smith H, Dowe N, Kalyuzhnaya MG, Pienkos PT, Guarnieri MT
  TITLE     Bioconversion of methane to lactate by an obligate methanotrophic 
            bacterium.
  JOURNAL   Sci Rep. 2016 Feb 23;6:21585. doi: 10.1038/srep21585.
  PUBMED    26902345
REFERENCE   2  (bases 1 to 7627)
  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: "Sci Rep."; 
            date: "2016-02-23"; volume: "6:21585. doi"; pages: " 
            10.1038/srep21585"
COMMENT     Sequence Label: pCAH01
FEATURES             Location/Qualifiers
     source          1..7627
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..105
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(106..174,175..966)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(324..343)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             973..1599
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(1049..1068)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             complement(1861..2676)
                     /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(1861..2676)
                     /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     2584..2603
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2668..2687
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             complement(2980..4128)
                     /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(4400..4771)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSL*AYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            4804..4913
                     /note="incP origin of transfer"
     rep_origin      5066..5697
                     /label=oriV
                     /note="incP origin of replication"
     rep_origin      6006..6594
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6495..6514
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        6805..6860
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    6811..6829
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     protein_bind    6841..6859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     sig_peptide     6908..6970
                     /codon_start=1
                     /product="signal peptide from the E. coli outer membrane 
                     protein OmpA"
                     /label=OmpA signal peptide
                     /translation="MKKTAIAIAVALAGFATVAQA"
     rep_origin      7146..7601
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7233..7252)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7443..7464
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
       61 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
      121 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
      181 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
      241 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
      301 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
      361 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
      421 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
      481 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
      541 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
      601 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
      661 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaattg
      721 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
      781 ggctggttta ttgctgataa atctggagcc ggtgagcgtg gctctcgcgg tatcattgca
      841 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
      901 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
      961 tggtaggaat taatgatgtc tcgtttagat aaaagtaaag tgattaacag cgcattagag
     1021 ctgcttaatg aggtcggaat cgaaggttta acaacccgta aactcgccca gaagctaggt
     1081 gtagagcagc ctacattgta ttggcatgta aaaaataagc gggctttgct cgacgcctta
     1141 gccattgaga tgttagatag gcaccatact cacttttgcc ctttagaagg ggaaagctgg
     1201 caagattttt tacgtaataa cgctaaaagt tttagatgtg ctttactaag tcatcgcgat
     1261 ggagcaaaag tacatttagg tacacggcct acagaaaaac agtatgaaac tctcgaaaat
     1321 caattagcct ttttatgcca acaaggtttt tcactagaga atgcattata tgcactcagc
     1381 gcagtggggc attttacttt aggttgcgta ttggaagatc aagagcatca agtcgctaaa
     1441 gaagaaaggg aaacacctac tactgatagt atgccgccat tattacgaca agctatcgaa
     1501 ttatttgatc accaaggtgc agagccagcc ttcttattcg gccttgaatt gatcatatgc
     1561 ggattagaaa aacaacttaa atgtgaaagt gggtcttaaa agcagcataa cctttttccg
     1621 tgatggtaac ttcactagtt taaaaggatc taggtgaaga tcctttttga taatctcatg
     1681 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaattgt
     1741 cgggaagatg cgtgatctga tccttcaact cagcaaaagt tcgatttatt caacaaagcc
     1801 gccgtcccgt caagtcagcg taatgctctg ccagtgttac aaccaattaa ccaattctga
     1861 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
     1921 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     1981 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     2041 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     2101 tgaatccggt gagaatggca aaagcttatg catttctttc cagacttgtt caacaggcca
     2161 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     2221 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
     2281 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     2341 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
     2401 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     2461 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     2521 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     2581 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     2641 cctcgagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat
     2701 gtaagcagac agttttattg ttcatgatga tatattttta tcttgtgcaa tgtaacatca
     2761 gagattttga gacacaacgt ggctttcgac ttccggcaag ctatacgcgc cctagaattg
     2821 tcaattttaa tcctctgttt atcggcagtt cgtagagcgc gccgtgcgtc ccgagcgata
     2881 ctgagcgaag caagtgcgtc gagcagtgcc cgcttgttcc tgaaatgcca gtaaagcgct
     2941 ggctgctgaa cccccagccg gaactgaccc cacaaggccc tagcgtttgc aatgcaccag
     3001 gtcatcattg acccaggcgt gttccaccag gccgctgcct cgcaactctt cgcaggcttc
     3061 gccgacctgc tcgcgccact tcttcacgcg ggtggaatcc gatccgcaca tgaggcggaa
     3121 ggtttccagc ttgagcgggt acggctcccg gtgcgagctg aaatagtcga acatccgtcg
     3181 ggccgtcggc gacagcttgc ggtacttctc ccatatgaat ttcgtgtagt ggtcgccagc
     3241 aaacagcacg acgatttcct cgtcgatcag gacctggcaa cgggacgttt tcttgccacg
     3301 gtccaggacg cggaagcggt gcagcagcga caccgattcc aggtgcccaa cgcggtcgga
     3361 cgtgaagccc atcgccgtcg cctgtaggcg cgacaggcat tcctcggcct tcgtgtaata
     3421 ccggccattg atcgaccagc ccaggtcctg gcaaagctcg tagaacgtga aggtgatcgg
     3481 ctcgccgata ggggtgcgct tcgcgtactc caacacctgc tgccacacca gttcgtcatc
     3541 gtcggcccgc agctcgacgc cggtgtaggt gatcttcacg tccttgttga cgtggaaaat
     3601 gaccttgttt tgcagcgcct cgcgcgggat tttcttgttg cgcgtggtga acagggcaga
     3661 gcgggccgtg tcgtttggca tcgctcgcat cgtgtccggc cacggcgcaa tatcgaacaa
     3721 ggaaagctgc atttccttga tctgctgctt cgtgtgtttc agcaacgcgg cctgcttggc
     3781 ctcgctgacc tgttttgcca ggtcctcgcc ggcggttttt cgcttcttgg tcgtcatagt
     3841 tcctcgcgtg tcgatggtca tcgacttcgc caaacctgcc gcctcctgtt cgagacgacg
     3901 cgaacgctcc acggcggccg atggcgcggg cagggcaggg ggagccagtt gcacgctgtc
     3961 gcgctcgatc ttggccgtag cttgctggac catcgagccg acggactgga aggtttcgcg
     4021 gggcgcacgc atgacggtgc ggcttgcgat ggtttcggca tcctcggcgg aaaaccccgc
     4081 gtcgatcagt tcttgcctgt atgccttccg gtcaaacgtc cgattcattc accctccttg
     4141 cgggattgcc ccgactcacg ccggggcaat gtgcccttat tcctgatttg acccgcctgg
     4201 tgccttggtg tccagataat ccaccttatc ggcaatgaag tcggtcccgt agaccgtctg
     4261 gccgtccttc tcgtacttgg tattccgaat cttgccctgc acgaatacca gctccgcgaa
     4321 gtcgctcttc ttgatggagc gcatggggac gtgcttggca atcacgcgca ccccccggcc
     4381 gttttagcgg ctaaaaaagt catggctctg ccctcgggcg gaccacgccc atcatgacct
     4441 tgccaagctc gtcctgcttc tcttcgatct tcgccagcag ggcgaggatc gtggcatcac
     4501 cgaaccgcgc cgtgcgcggg tcgtcggtga gccagagttt cagcaggccg cccaggcggc
     4561 ccaggtcgcc attgatgcgg gccagctcgc ggacgtgctc atagtccacg acgcccgtga
     4621 ttttgtagcc ctggccgacg gccagcaggt aggcctacag gctcatgccg gccgccgccg
     4681 ccttttcctc aatcgctctt cgttcgtctg gaaggcagta caccttgata ggtgggctgc
     4741 ccttcctggt tggcttggtt tcatcagcca tccgcttgcc ctcatctgtt acgccggcgg
     4801 tagccggcca gcctcgcaga gcaggattcc cgttgagcac cgccaggtgc gaataaggga
     4861 cagtgaagaa ggaacacccg ctcgcgggtg ggcctacttc acctatcctg cccggctgac
     4921 gccgttggat acaccaagga aagtctacac gaaccctttg gcaaaatcct gtatatcgtg
     4981 cgaaaaagga tggatatacc gaaaaaatcg ctataatgac cccgaagcag ggttatgcag
     5041 cggaaaagat ccgtcgaccc tttccgacgc tcaccgggct ggttgccctc gccgctgggc
     5101 tggcggccgt ctatggccct gcaaacgcgc cagaaacgcc gtcgaagccg tgtgcgagac
     5161 accgcggccg ccggcgttgt ggatacctcg cggaaaactt ggccctcact gacagatgag
     5221 gggcggacgt tgacacttga ggggccgact cacccggcgc ggcgttgaca gatgaggggc
     5281 aggctcgatt tcggccggcg acgtggagct ggccagcctc gcaaatcggc gaaaacgcct
     5341 gattttacgc gagtttccca cagatgatgt ggacaagcct ggggataagt gccctgcggt
     5401 attgacactt gaggggcgcg actactgaca gatgaggggc gcgatccttg acacttgagg
     5461 ggcagagtgc tgacagatga ggggcgcacc tattgacatt tgaggggctg tccacaggca
     5521 gaaaatccag catttgcaag ggtttccgcc cgtttttcgg ccaccgctaa cctgtctttt
     5581 aacctgcttt taaaccaata tttataaacc ttgtttttaa ccagggctgc gccctgtgcg
     5641 cgtgaccgcg cacgccgaag gggggtgccc ccccttctcg aaccctcccg gcccgctaac
     5701 gcgggcctcc catcccccca ggggctgcgc ccctcggccg cgaacggcct caccccaaaa
     5761 atggcagcca agctgacccg ctagggacgt gaagtcgatt ccttcgatgg ttagcaatca
     5821 aagactcaga gtgctgtctg gagcgtgaat ctaacggtac gtatctcgat tgctcggtcg
     5881 ctattcgcac tctgcgaaag ttcgtaccgc tcattcacta ggttgcgaat catgaccaaa
     5941 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6001 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6061 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6121 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     6181 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6241 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6301 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6361 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6421 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     6481 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     6541 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     6601 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     6661 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgggcct
     6721 ttttacggtt cctggccttt tgctggcctt ttgctcacat gacccgacac catcgaatgg
     6781 ccagatgatt aattcctaat ttttgttgac actctatcat tgatagagtt attttaccac
     6841 tccctatcag tgatagagaa aagtgaaatg aatagttcga caaaaatcta gataacgagg
     6901 gcaaaaaatg aaaaagacag ctatcgcgat tgcagtggca ctggctggtt tcgctaccgt
     6961 agcgcaggcc tgagaccaga attcgagctc ggtacccggg gatccctcga ggtcgacctg
     7021 caggcagcgc ttggcgtcac ccgcagttcg gtggttaata agcttgacct gtgaagtgaa
     7081 aaatggcgca cattgtgcga catttttttt gtctgccgtt taccgctact gcgtcacgga
     7141 tctccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     7201 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     7261 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     7321 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     7381 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     7441 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     7501 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     7561 atttaacgcg aattttaaca aaatattaac gcttacaatt tcaggtggca cttttcgggg
     7621 aaatgtg
//
