LOCUS       sequence-207090-        8445 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Broad host range plasmid for constitutive expression of the eGFP. 
            Developed for imaging Pseudomonas aeruginosa in biofilms..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8445)
  AUTHORS   Nivens DE, Ohman DE, Williams J, Franklin MJ
  TITLE     Role of alginate and its O acetylation in formation of Pseudomonas 
            aeruginosa microcolonies and biofilms.
  JOURNAL   J Bacteriol. 2001 Feb;183(3):1047-57.
  PUBMED    11208804
REFERENCE   2  (bases 1 to 8445)
  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: "J 
            Bacteriol."; date: "2001-02"; volume: "183(3)"; pages: "1047-57"
COMMENT     Sequence Label: pMF230
FEATURES             Location/Qualifiers
     source          1..8445
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..23,24..30)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    38..54
                     /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     43..65
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             110..823
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFAYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(140..168)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     763..783
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(863..880)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1033..1119
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1211..1238
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1257..1348
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1349..1417,1418..2209)
                     /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(1567..1586)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2380..2968
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2869..2888
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3122..3139
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3154..3294
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3220..3239)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3380..3402
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3396..3587)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5045..5064
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     oriT            5494..5603
                     /note="incP origin of transfer"
     CDS             complement(6373..7206)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pRO1600 from Pseudomonas aeruginosa "
                     /label=pRO1600 Rep
                     /translation="MASPPMVYKSNALVEAAYRLSVQEQRIVLACISQVKRSEPVTDEV
                     MYSVTAEDIATMAGVPIESSYNQLKEAALRLKRREVRLTQEPNGKGKRPSVMITGWVQT
                     IIYREGEGRVELRFTKDMLPYLTELTKQFTKYALADVAKMDSTHAIRLYELLMQWDSIG
                     QREIEIDQLRKWFQLEGRYPSIKDFKLRVLDPAVTQINEHSPLQVEWAQRKTGRKVTHL
                     LFSFGPKKPAKAVGKAPAKRKAGKISDAEIAKQARPGETWEAARARLTQMPLDLA"
     rep_origin      7220..7571
                     /label=pRO1600 oriV
                     /note="broad-host-range origin of replication from 
                     Pseudomonas aeruginosa plasmid pRO1600; requires the 
                     pRO1600 Rep protein for replication (West et al., 1994)"
ORIGIN
        1 ttgacaatta atcatccggc tcgtataatg tgtggaattg tgagcggata acaatttcac
       61 acaggaaaca gaccatggct gcagtctaga gtcgacagga gaagaaaaaa tgagtaaagg
      121 agaagaactt ttcactggag ttgtcccaat tcttgttgaa ttagatggtg atgttaatgg
      181 gcacaaattt tctgtcagtg gagagggtga aggtgatgca acatacggaa aacttaccct
      241 taaatttatt tgcactactg gaaaactacc tgttccatgg ccaacacttg tcactacttt
      301 cgcgtatggt cttcaatgct ttgcgagata cccagatcat atgaaacagc atgacttttt
      361 caagagtgcc atgcccgaag gttatgtaca ggaaagaact atatttttca aagatgacgg
      421 gaactacaag acacgtgctg aagtcaagtt tgaaggtgat acccttgtta atagaatcga
      481 gttaaaaggt attgatttta aagaagatgg aaacattctt ggacacaaat tggaatacaa
      541 ctataactca cacaatgtat acatcatggc agacaaacaa aagaatggaa tcaaagttaa
      601 cttcaaaatt agacacaaca ttgaagatgg aagcgttcaa ctagcagacc attatcaaca
      661 aaatactcca attggcgatg gccctgtcct tttaccagac aaccattacc tgtccacaca
      721 atctgccctt tcgaaagatc ccaacgaaaa gagagaccac atggtccttc ttgagtttgt
      781 aacagctgct gggattacac atggcatgga tgaactatac aaataagctt ggctgttttg
      841 gcggatgaga gaagattttc agcctgatac agattaaatc agaacgcaga agcggtctga
      901 taaaacagaa tttgcctggc ggcagtagcg cggtggtccc acctgacccc atgccgaact
      961 cagaagtgaa acgccgtagc gccgatggta gtgtggggtc tccccatgcg agagtaggga
     1021 actgccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc
     1081 tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccgggagc ggatttgaac
     1141 gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggcat
     1201 caaattaagc agaaggccat cctgacggat ggcctttttg cgtttctaca aactcttttg
     1261 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     1321 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     1381 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     1441 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     1501 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     1561 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg
     1621 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     1681 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     1741 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     1801 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     1861 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     1921 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     1981 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     2041 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     2101 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     2161 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     2221 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     2281 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     2341 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     2401 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     2461 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     2521 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     2581 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     2641 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     2701 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     2761 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     2821 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     2881 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     2941 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     3001 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     3061 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     3121 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     3181 tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc
     3241 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     3301 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     3361 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     3421 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     3481 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     3541 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     3601 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     3661 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     3721 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     3781 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     3841 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     3901 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     3961 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     4021 ctcaacgaca ggagcacgat catgcgcacc cgtggccagg acccaacgct gcccgagatg
     4081 cgccgcgtgc ggctgctgga gatggcggac gcgatggata tgttctgcca agggttggtt
     4141 tgcgcattca cagttctccg caagaattga ttggctccaa ttcttggagt ggtgaatccg
     4201 ttagcgaggt gccgccggct tccattcagg tcgaggtggc ccggctccat gcaccgcgac
     4261 gcaacgcggg gaggcagaca aggtataggg cggcgcctac aatccatgcc aacccgttcc
     4321 atgtgctcgc cgaggcggca taaatcgccg tgacgatcag cggtccaatg atcgaagtta
     4381 ggctggtaag agccgcgagc gatccttgaa gctgtccctg atggtcgtca tctacctgcc
     4441 tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa
     4501 tggggaaggc catccagcct cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg
     4561 ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga
     4621 cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg
     4681 tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc
     4741 ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg
     4801 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga cgctctccct
     4861 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     4921 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     4981 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     5041 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     5101 ccacgatgcg tccggcgtag aggatcttcc atacctacca gttctgcgcc tgcagcaatg
     5161 gcaacaacgt tgcccggatc gtggcatcac cgaaccgcgc cgtgcgcggg tcgtcggtga
     5221 gccagagttt cagcaggccg cccaggcggc ccaggtcgcc attgatgcgg gccagctcgc
     5281 ggacgtgctc atagtccacg acgcccgtga ttttgtagcc ctggccgacg gccagcaggt
     5341 aggccgacag gctcatgccg gccgccgccg ccttttcctc aatcgctctt cgttcgtctg
     5401 gaaggcagta caccttgata ggtgggctgc ccttcctggt tggcttggtt tcatcagcca
     5461 tccgcttgcc ctcatctgtt acgccggcgg tagccggcca gcctcgcaga gcaggattcc
     5521 cgttgagcac cgccaggtgc gaataaggga cagtgaagaa ggaacacccg ctcgcgggtg
     5581 ggcctacttc acctatcctg cccggctgac gccgttggat acaccaagga aagtctacac
     5641 gaaccctttg gcaaaatcct gtatatcgtg cgaaaaagga tggatatacc gaaaaaatcg
     5701 ctataatgac cccgaagcag ggttatgcag cggaaaagcg ctgcttccct gctgttttgt
     5761 ggaatatcta ccgactggaa acaggcaaat gcaggaaatt actgaactga ggggacaggc
     5821 gagagacgat gccaaagagc tacaccgacg agctggccga gtgggttgaa tcccgcgcgg
     5881 ccaagaagcg ccggcgtgat gaggctgcgg ttgcgttcct ggcggtgagg gcggatgtcg
     5941 aggcggcgtt agcgtccggc tatgcgctcg tcaccatttg ggagcacatg cgggaaacgg
     6001 ggaaggtcaa gttctcctac gagacgttcc gctcgcacgc caggcggcac atcaaggcca
     6061 agcccgccga tgtgcccgca ccgcaggcca aggctgcgga acccgcgccg gcacccaaga
     6121 cgccggagcc acggcggccg aagcaggggg gcaaggctga aaagccggcc cccgctgcgg
     6181 ccccgaccgg cttcaccttc aacccaacac cggacaaaaa ggatcggcgt tgccatagtc
     6241 gattttcttc aaatagcgcc gagctttgtc atcaccaatt tcctttgcaa atagctcata
     6301 cacccaaaca gtagaattcc ccggatccgt cgacctgcag aaaggcaggc cgggccgtgg
     6361 tggccacggc ctctaggcca gatccagcgg catctgggtt agtcgagcgc gggccgcttc
     6421 ccatgtctca ccagggcgag cctgtttcgc gatctcagca tctgaaatct tcccggcctt
     6481 gcgcttcgct ggggccttac ccaccgcctt ggcgggcttc ttcggtccaa aactgaacaa
     6541 cagatgtgtg accttgcgcc cggtctttcg ctgcgcccac tccacctgta gcgggctgtg
     6601 ctcgttgatc tgcgtcacgg ctggatcaag cactcgcaac ttgaagtcct tgatcgaggg
     6661 ataccggcct tccagttgaa accactttcg cagctggtca atttctattt cgcgctggcc
     6721 gatgctgtcc cattgcatga gcagctcgta aagcctgatc gcgtgggtgc tgtccatctt
     6781 ggccacgtca gccaaggcgt atttggtgaa ctgtttggtg agttccgtca ggtacggcag
     6841 catgtctttg gtgaacctga gttctacacg gccctcaccc tcccggtaga tgattgtttg
     6901 cacccagccg gtaatcatca cactcggtct tttccccttg ccattgggct cttgggttaa
     6961 ccggacttcc cgccgtttca ggcgcagggc cgcttctttg agctggttgt aggaagattc
     7021 gatagggaca cccgccatcg tcgctatgtc ctccgccgtc actgaataca tcacttcatc
     7081 ggtgacaggc tcgctcctct tcacctggct aatacaggcc agaacgatcc gctgttcctg
     7141 aacactgagg cgatacgcgg cctcgaccag ggcattgctt ttgtaaacca ttgggggtga
     7201 ggccacgttc gacattcctt gtgtataagg ggacactgta tctgcgtccc acaatacaac
     7261 aaatccgtcc ctttacaaca acaaatccgt cccttcttaa caacaaatcc gtcccttaat
     7321 ggcaacaaat ccgtcccttt ttaaactcta caggccacgg attacgtggc ctgtagacgt
     7381 cctaaaaggt ttaaaaggga aaaggaagaa aagggtggaa acgcaaaaaa cgcaccacta
     7441 cgtggccccg ttggggccgc atttgtgccc ctgaaggggc gggggaggcg tctgggcaat
     7501 ccccgtttta ccagtcccct atcgccgcct gagagggcgc aggaagcgag taatcagggt
     7561 atcgaggcgg attcaccctt ggcgtccaac cagcggcacc agcggcgcct gagaggccta
     7621 cagagcggtt ggacaccaag gggaggggct aagaccggtt tatcagtccc ctttccctcg
     7681 tttctttcca acgcgatagc ccagcaaggc cgccaccgtt gccaccgtca ccccagcaag
     7741 cacagccagt ggcgtgtaat tggggtctga cgctcagtgg aacgaaaact cacgttaagg
     7801 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     7861 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     7921 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     7981 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     8041 gataccgcga gatccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     8101 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     8161 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     8221 tgctgcaggt cgacggatcc ggggaattct catgtttgac agcttatcat cgactgcacg
     8281 gtgcaccaat gcttctggcg tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc
     8341 gtaaatcact gcataattcg tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt
     8401 gcgccgacat cataacggtt ctggcaaata ttctgaaatg agctg
//
