LOCUS       sequence-245521-        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"
     CDS             561..1187
                     /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(637..656)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             complement(1449..2264)
                     /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(1449..2264)
                     /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     2172..2191
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2256..2275
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             complement(2568..3716)
                     /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(3988..4359)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSL*AYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            4392..4501
                     /note="incP origin of transfer"
     rep_origin      4654..5285
                     /label=oriV
                     /note="incP origin of replication"
     rep_origin      5594..6182
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6083..6102
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        6393..6448
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    6399..6417
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     protein_bind    6429..6447
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     sig_peptide     6496..6558
                     /codon_start=1
                     /product="signal peptide from the E. coli outer membrane 
                     protein OmpA"
                     /label=OmpA signal peptide
                     /translation="MKKTAIAIAVALAGFATVAQA"
     rep_origin      6734..7189
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6821..6840)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7031..7052
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        7216..7320
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7321..7389,7390..7627,1..554)
                     /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(7539..7558)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
       61 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
      121 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
      181 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
      241 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
      301 aacaattgat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
      361 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggc tctcgcggta
      421 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
      481 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
      541 ttaagcattg gtaggaatta atgatgtctc gtttagataa aagtaaagtg attaacagcg
      601 cattagagct gcttaatgag gtcggaatcg aaggtttaac aacccgtaaa ctcgcccaga
      661 agctaggtgt agagcagcct acattgtatt ggcatgtaaa aaataagcgg gctttgctcg
      721 acgccttagc cattgagatg ttagataggc accatactca cttttgccct ttagaagggg
      781 aaagctggca agatttttta cgtaataacg ctaaaagttt tagatgtgct ttactaagtc
      841 atcgcgatgg agcaaaagta catttaggta cacggcctac agaaaaacag tatgaaactc
      901 tcgaaaatca attagccttt ttatgccaac aaggtttttc actagagaat gcattatatg
      961 cactcagcgc agtggggcat tttactttag gttgcgtatt ggaagatcaa gagcatcaag
     1021 tcgctaaaga agaaagggaa acacctacta ctgatagtat gccgccatta ttacgacaag
     1081 ctatcgaatt atttgatcac caaggtgcag agccagcctt cttattcggc cttgaattga
     1141 tcatatgcgg attagaaaaa caacttaaat gtgaaagtgg gtcttaaaag cagcataacc
     1201 tttttccgtg atggtaactt cactagttta aaaggatcta ggtgaagatc ctttttgata
     1261 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     1321 aaaattgtcg ggaagatgcg tgatctgatc cttcaactca gcaaaagttc gatttattca
     1381 acaaagccgc cgtcccgtca agtcagcgta atgctctgcc agtgttacaa ccaattaacc
     1441 aattctgatt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     1501 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     1561 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     1621 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     1681 gtgacgactg aatccggtga gaatggcaaa agcttatgca tttctttcca gacttgttca
     1741 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     1801 cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca
     1861 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     1921 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     1981 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     2041 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     2101 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     2161 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     2221 aatcgcggcc tcgagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta
     2281 ctgtttatgt aagcagacag ttttattgtt catgatgata tatttttatc ttgtgcaatg
     2341 taacatcaga gattttgaga cacaacgtgg ctttcgactt ccggcaagct atacgcgccc
     2401 tagaattgtc aattttaatc ctctgtttat cggcagttcg tagagcgcgc cgtgcgtccc
     2461 gagcgatact gagcgaagca agtgcgtcga gcagtgcccg cttgttcctg aaatgccagt
     2521 aaagcgctgg ctgctgaacc cccagccgga actgacccca caaggcccta gcgtttgcaa
     2581 tgcaccaggt catcattgac ccaggcgtgt tccaccaggc cgctgcctcg caactcttcg
     2641 caggcttcgc cgacctgctc gcgccacttc ttcacgcggg tggaatccga tccgcacatg
     2701 aggcggaagg tttccagctt gagcgggtac ggctcccggt gcgagctgaa atagtcgaac
     2761 atccgtcggg ccgtcggcga cagcttgcgg tacttctccc atatgaattt cgtgtagtgg
     2821 tcgccagcaa acagcacgac gatttcctcg tcgatcagga cctggcaacg ggacgttttc
     2881 ttgccacggt ccaggacgcg gaagcggtgc agcagcgaca ccgattccag gtgcccaacg
     2941 cggtcggacg tgaagcccat cgccgtcgcc tgtaggcgcg acaggcattc ctcggccttc
     3001 gtgtaatacc ggccattgat cgaccagccc aggtcctggc aaagctcgta gaacgtgaag
     3061 gtgatcggct cgccgatagg ggtgcgcttc gcgtactcca acacctgctg ccacaccagt
     3121 tcgtcatcgt cggcccgcag ctcgacgccg gtgtaggtga tcttcacgtc cttgttgacg
     3181 tggaaaatga ccttgttttg cagcgcctcg cgcgggattt tcttgttgcg cgtggtgaac
     3241 agggcagagc gggccgtgtc gtttggcatc gctcgcatcg tgtccggcca cggcgcaata
     3301 tcgaacaagg aaagctgcat ttccttgatc tgctgcttcg tgtgtttcag caacgcggcc
     3361 tgcttggcct cgctgacctg ttttgccagg tcctcgccgg cggtttttcg cttcttggtc
     3421 gtcatagttc ctcgcgtgtc gatggtcatc gacttcgcca aacctgccgc ctcctgttcg
     3481 agacgacgcg aacgctccac ggcggccgat ggcgcgggca gggcaggggg agccagttgc
     3541 acgctgtcgc gctcgatctt ggccgtagct tgctggacca tcgagccgac ggactggaag
     3601 gtttcgcggg gcgcacgcat gacggtgcgg cttgcgatgg tttcggcatc ctcggcggaa
     3661 aaccccgcgt cgatcagttc ttgcctgtat gccttccggt caaacgtccg attcattcac
     3721 cctccttgcg ggattgcccc gactcacgcc ggggcaatgt gcccttattc ctgatttgac
     3781 ccgcctggtg ccttggtgtc cagataatcc accttatcgg caatgaagtc ggtcccgtag
     3841 accgtctggc cgtccttctc gtacttggta ttccgaatct tgccctgcac gaataccagc
     3901 tccgcgaagt cgctcttctt gatggagcgc atggggacgt gcttggcaat cacgcgcacc
     3961 ccccggccgt tttagcggct aaaaaagtca tggctctgcc ctcgggcgga ccacgcccat
     4021 catgaccttg ccaagctcgt cctgcttctc ttcgatcttc gccagcaggg cgaggatcgt
     4081 ggcatcaccg aaccgcgccg tgcgcgggtc gtcggtgagc cagagtttca gcaggccgcc
     4141 caggcggccc aggtcgccat tgatgcgggc cagctcgcgg acgtgctcat agtccacgac
     4201 gcccgtgatt ttgtagccct ggccgacggc cagcaggtag gcctacaggc tcatgccggc
     4261 cgccgccgcc ttttcctcaa tcgctcttcg ttcgtctgga aggcagtaca ccttgatagg
     4321 tgggctgccc ttcctggttg gcttggtttc atcagccatc cgcttgccct catctgttac
     4381 gccggcggta gccggccagc ctcgcagagc aggattcccg ttgagcaccg ccaggtgcga
     4441 ataagggaca gtgaagaagg aacacccgct cgcgggtggg cctacttcac ctatcctgcc
     4501 cggctgacgc cgttggatac accaaggaaa gtctacacga accctttggc aaaatcctgt
     4561 atatcgtgcg aaaaaggatg gatataccga aaaaatcgct ataatgaccc cgaagcaggg
     4621 ttatgcagcg gaaaagatcc gtcgaccctt tccgacgctc accgggctgg ttgccctcgc
     4681 cgctgggctg gcggccgtct atggccctgc aaacgcgcca gaaacgccgt cgaagccgtg
     4741 tgcgagacac cgcggccgcc ggcgttgtgg atacctcgcg gaaaacttgg ccctcactga
     4801 cagatgaggg gcggacgttg acacttgagg ggccgactca cccggcgcgg cgttgacaga
     4861 tgaggggcag gctcgatttc ggccggcgac gtggagctgg ccagcctcgc aaatcggcga
     4921 aaacgcctga ttttacgcga gtttcccaca gatgatgtgg acaagcctgg ggataagtgc
     4981 cctgcggtat tgacacttga ggggcgcgac tactgacaga tgaggggcgc gatccttgac
     5041 acttgagggg cagagtgctg acagatgagg ggcgcaccta ttgacatttg aggggctgtc
     5101 cacaggcaga aaatccagca tttgcaaggg tttccgcccg tttttcggcc accgctaacc
     5161 tgtcttttaa cctgctttta aaccaatatt tataaacctt gtttttaacc agggctgcgc
     5221 cctgtgcgcg tgaccgcgca cgccgaaggg gggtgccccc ccttctcgaa ccctcccggc
     5281 ccgctaacgc gggcctccca tccccccagg ggctgcgccc ctcggccgcg aacggcctca
     5341 ccccaaaaat ggcagccaag ctgacccgct agggacgtga agtcgattcc ttcgatggtt
     5401 agcaatcaaa gactcagagt gctgtctgga gcgtgaatct aacggtacgt atctcgattg
     5461 ctcggtcgct attcgcactc cgcgaaagtt cgtaccgctc attcactagg ttgcgaatca
     5521 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     5581 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     5641 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     5701 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     5761 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     5821 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     5881 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     5941 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     6001 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     6061 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     6121 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     6181 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     6241 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     6301 ctgggccttt ttacggttcc tggccttttg ctggcctttt gctcacatga cccgacacca
     6361 tcgaatggcc agatgattaa ttcctaattt ttgttgacac tctatcattg atagagttat
     6421 tttaccactc cctatcagtg atagagaaaa gtgaaatgaa tagttcgaca aaaatctaga
     6481 taacgagggc aaaaaatgaa aaagacagct atcgcgattg cagtggcact ggctggtttc
     6541 gctaccgtag cgcaggcctg agaccagaat tcgagctcgg tacccgggga tccctcgagg
     6601 tcgacctgca ggcagcgctt ggcgtcaccc gcagttcggt ggttaataag cttgacctgt
     6661 gaagtgaaaa atggcgcaca ttgtgcgaca ttttttttgt ctgccgttta ccgctactgc
     6721 gtcacggatc tccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     6781 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     6841 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     6901 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     6961 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     7021 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     7081 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     7141 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc aggtggcact
     7201 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     7261 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     7321 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     7381 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     7441 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     7501 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     7561 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     7621 gttgagt
//
