LOCUS       sequence-333399-        6800 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Suicide vector for integrating expression cassette in P. putida 
            (L-rhamnose induction).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6800)
  AUTHORS   Cook TB, Jacobson TB, Venkataraman MV, Hofstetter H, Amador-Noguez 
            D, Thomas MG, Pfleger BF
  TITLE     Stepwise genetic engineering of Pseudomonas putida enables robust 
            heterologous production of prodigiosin and glidobactin A.
  JOURNAL   Metab Eng. 2021 Jun 24;67:112-124. doi: 10.1016/j.ymben.2021.06.004.
  PUBMED    34175462
REFERENCE   2  (bases 1 to 6800)
  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: "Metab 
            Eng."; date: "2021-06-24"; volume: "67:112-124. doi"; pages: " 
            10.1016/j.ymben.2021.06.004"
COMMENT     Sequence Label: pNVLTv2-pvdIJD-Prha-base
FEATURES             Location/Qualifiers
     source          1..6800
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1..849)
                     /codon_start=1
                     /gene="E. coli rhaR"
                     /product="transcriptional activator of rhaR and rhaS"
                     /label=rhaR
                     /note="needed for rhamnose-dependent activation of the rhaB
                     promoter"
                     /translation="MAHQLKLLKDDFFASDQQAVAVADRYPQDVFAEHTHDFCELVIVW
                     RGNGLHVLNDRPYRITRGDLFYIHADDKHSYASVNDLVLQNIIYCPERLKLNLDWQGAI
                     PGFNASAGQPHWRLGSMGMAQARQVIGQLEHESSQHVPFANEMAELLFGQLVMLLNRHR
                     YTSDSLPPTSSETLLDKLITRLAASLKSPFALDKFCDEASCSERVLRQQFRQQTGMTIN
                     QYLRQVRVCHAQYLLQHSRLLISDISTECGFEDSNYFSVVFTRETGMTPSQWRHLNSQK
                     D"
     CDS             complement(923..1759)
                     /codon_start=1
                     /gene="E. coli rhaS"
                     /product="positive regulator of the rhaB promoter
                     "
                     /label=rhaS
                     /note="activated by L-rhamnose"
                     /translation="MTVLHSVDFFPSGNASVAIEPRLPQADFPEHHHDFHEIVIVEHGT
                     GIHVFNGQPYTITGGTVCFVRDHDRHLYEHTDNLCLTNVLYRSPDRFQFLAGLNQLLPQ
                     ELDGQYPSHWRVNHSVLQQVRQLVAQMEQQEGENDLPSTASREILFMQLLLLLRKSSLQ
                     ENLENSASRLNLLLAWLEDHFADEVNWDAVADQFSLSLRTLHRQLKQQTGLTPQRYLNR
                     LRLMKARHLLRHSEASVTDIAYRCGFSDSNHFSTLFRREFNWSPRDIRQGRDGFLQ"
     promoter        1905..2023
                     /label=rhaB promoter
                     /note="promoter of the E. coli rhaBAD operon, conferring 
                     tight induction with L-rhamnose and repression with 
                     D-glucose in the presence of RhaR and RhaS (Giacalone et 
                     al., 2006)"
     primer_bind     complement(3187..3204)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3321..3866)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(3401..3420)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3942..3961
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     oriT            4089..4197
                     /note="incP origin of transfer"
     primer_bind     4227..4245
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             4552..5346
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4606..4625)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5216..5235
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 ttaatctttc tgcgaattga gatgacgcca ctggctgggc gtcatcccgg tttcccgggt
       61 aaacaccacc gaaaaatagt tactatcttc aaagccacat tcggtcgaaa tatcactgat
      121 taacaggcgg ctatgctgga gaagatattg cgcatgacac actctgacct gtcgcagata
      181 ttgattgatg gtcattccag tctgctggcg aaattgctga cgcaaaacgc gctcactgca
      241 cgatgcctca tcacaaaatt tatccagcgc aaagggactt ttcaggctag ccgccagccg
      301 ggtaatcagc ttatccagca acgtttcgct ggatgttggc ggcaacgaat cactggtgta
      361 acgatggcga ttcagcaaca tcaccaactg cccgaacagc aactcagcca tttcgttagc
      421 aaacggcaca tgctgactac tttcatgctc aagctgaccg ataacctgcc gcgcctgcgc
      481 catccccatg ctacctaagc gccagtgtgg ttgccctgcg ctggcgttaa atcccggaat
      541 cgccccctgc cagtcaagat tcagcttcag acgctccggg caataaataa tattctgcaa
      601 aaccagatcg ttaacggaag cgtaggagtg tttatcgtca gcatgaatgt aaaagagatc
      661 gccacgggta atgcgataag ggcgatcgtt gagtacatgc aggccattac cgcgccagac
      721 aatcaccagc tcacaaaaat catgtgtatg ttcagcaaag acatcttgcg gataacggtc
      781 agccacagcg actgcctgct ggtcgctggc aaaaaaatca tctttgagaa gttttaactg
      841 atgcgccacc gtggctacct cggccagaga acgaagttga ttattcgcaa tatggcgtac
      901 aaatacgttg agaagattcg cgttattgca gaaagccatc ccgtccctgg cgaatatcac
      961 gcggtgacca gttaaactct cggcgaaaaa gcgtcgaaaa gtggttactg tcgctgaatc
     1021 cacagcgata ggcgatgtca gtaacgctgg cctcgctgtg gcgtagcaga tgtcgggctt
     1081 tcatcagtcg caggcggttc aggtatcgct gaggcgtcag tcccgtttgc tgcttaagct
     1141 gccgatgtag cgtacgcagt gaaagagaaa attgatccgc cacggcatcc caattcacct
     1201 catcggcaaa atggtcctcc agccaggcca gaagcaagtt gagacgtgat gcgctgtttt
     1261 ccaggttctc ctgcaaactg cttttacgca gcaagagcag taattgcata aacaagatct
     1321 cgcgactggc ggtcgagggt aaatcatttt ccccttcctg ctgttccatc tgtgcaacca
     1381 gctgtcgcac ctgctgcaat acgctgtggt taacgcgcca gtgagacgga tactgcccat
     1441 ccagctcttg tggcagcaac tgattcagcc cggcgagaaa ctgaaatcga tccggcgagc
     1501 gatacagcac attggtcaga cacagattat cggtatgttc atacagatgc cgatcatgat
     1561 cgcgtacgaa acagaccgtg ccaccggtga tggtataggg ctgcccatta aacacatgaa
     1621 tacccgtgcc atgttcgaca atcacaattt catgaaaatc atgatgatgt tcaggaaaat
     1681 ccgcctgcgg gagccggggt tctatcgcca cggacgcgtt accagacgga aaaaaatcca
     1741 cactatgtaa tacggtcata ctggcctcct gatgtcgtca acacggcgaa atagtaatca
     1801 cgaggtcagg ttcttacctt aaattttcga cggaaaacca cgtaaaaaac gtcgattttt
     1861 caagatacag cgtgaatttt caggaaatgc ggtgagcatc acatcaccac aattcagcaa
     1921 attgtgaaca tcatcacgtt catctttccc tggttgccaa tggcccattt tcctgtcagt
     1981 aacgagaagg tcgcgaattc aggcgctctt tagactggtc gtaggtaccc tcgagtaagg
     2041 atctccagac gacgtacaag cgctgctcga acccaacctc acgcacgcct gcttcattga
     2101 actttcatga gctttcgggg ccgccagcgc ggcccctctg gagaatcgaa cgatgtccct
     2161 gaaccctgac ctggcggcct acctgcaact ggtggaggcc ggacgcagcg ccggcaaggt
     2221 gctgcccatg catgccctgg cggcggacga ggcgcgccgc cagttcgaag agtcctcggc
     2281 gctgattgcg ggcaaggccg acgagccgga ctgcatcagc gacctttcgt tgaccacccg
     2341 cgatggccat accttgccgg tgcgcttgta ccgcccgccg caggccgacc ctgcactggc
     2401 cggcgcggcg ctgctgtacc tgcatggcgg cggctacgtg gtgggcagcc tcgattcgca
     2461 tgacacgctg tgctggaacc tggcgcagga tgccggggtg ccggtgattg ccgtgggcta
     2521 ccgactggcg ccgcagtggc gtttcccgac agccagtgac gatgccctgg atgcctggcg
     2581 ctggttggtc gagcaggccg aggcgctggg catcgatgcg cagcgactgg cggtggtggg
     2641 cgacagtgtc ggtggtagcc tggctaccat tctggccaac cagttggctg cgcagcgcga
     2701 gctgccggca ccgcgcctgc aggtaatgat ctacccggtg accgatgcca gttgccggcg
     2761 gccttcggtg cagcgctatg gcagcggtta cctgcttgaa gcgcagaccc tggagtggtt
     2821 ctaccagcaa tacgccacgg tgccggcaga ccgtcacgat ccgcgttttt caccgttgct
     2881 gggcagcgtc gccagcaaca gtgcacctgc actgatgctg attgccgagt gcgacccgct
     2941 gcatgaccag ggtgtcgctt atgcacggca cttggagcaa gccggggtgg cggtgcagct
     3001 tgcggtgatc ccgggagtga cccatgactt catgcgtatg ggttcgatca tcgaggaggc
     3061 cgatgaaggc ctggcgatgg tggtcgaggc actgcagcag catctgtaag ggagcctttc
     3121 aggcatagct gtttcctgtg tgaaattgcg tcagcagaat atgtgataca ggatatattc
     3181 cgcttcctcg ctcactgact cgctacgctc ggtcgttcga ctgcggcgag cggaaatggc
     3241 ttacgaacgg ggcggagatt tcctggaaga tgccaggaag atacttaaca gggaagtgag
     3301 agggccgcgg caaagccgtt tttccatagg ctccgccccc ctgacaagca tcacgaaatc
     3361 tgacgctcaa atcagtggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     3421 cctggcggct ccctcgtgcg ctctcctgtt cctgcctttc ggtttaccgg tgtcattccg
     3481 ctgttatggc cgcgtttgtc tcattccacg cctgacactc agttccgggt aggcagttcg
     3541 ctccaagctg gactgtatgc acgaaccccc cgttcagtcc gaccgctgcg ccttatccgg
     3601 taactatcgt cttgagtcca acccggaaag acatgcaaaa gcaccactgg cagcagccac
     3661 tggtaattga tttagaggag ttagtcttga agtcatgcgc cggttaaggc taaactgaaa
     3721 ggacaagttt tggtgactgc gctcctccaa gccagttacc tcggttcaaa gagttggtag
     3781 ctcagagaac cttcgaaaaa ccgccctgca aggcggtttt ttcgttttca gagcaagaga
     3841 ttacgcgcag accaaaacga tctcaagaag atcatcttat taatcagata aaatatttct
     3901 aggctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
     3961 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
     4021 atctgctcat gtttgacagc ttatcgctca gtggaacgaa aactcgcatg tatccaacgg
     4081 cgtcagccgg gcaggatagg tgaagtaggc ccacccgcga gcgggtgttc cttcttcact
     4141 gtcccttatt cgcacctggc ggtgctcaac gggaatcctg ctctgcgagg ctggccgtag
     4201 tgcttggatt ctcaccaata tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc
     4261 gtttcggtga tgacggtgaa aacctctgag atgaatgtca gctactgggc tatctggaca
     4321 agggaaaacg caagcgcaaa gagaaagcag gtagcttgca gtgggcttac atggcgatag
     4381 ctagactggg cggttttatg gacagcaagc gaaccggaat tgccagctgg ggcgccctct
     4441 ggtaaggttg ggaagccctg caaagtaaac tggatggctt tcttgccgcc aaggatctga
     4501 tggcgcaggg gatcaagatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     4561 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     4621 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     4681 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     4741 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     4801 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     4861 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     4921 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     4981 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     5041 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     5101 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     5161 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     5221 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     5281 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     5341 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgtcatcac
     5401 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     5461 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccaccccg
     5521 ggctcgatcc cctcgcgagt tggttcagct gctgcctgag gctggacgac ctcgcggagt
     5581 tctaccggca gtgcaaatcc gtcggcatcc aggaaaccag cagcggctat ccgcgcatcc
     5641 atgcccccga actgcaggag tggggaggca cgatggccgc tttggtcccg gatcctctag
     5701 agtcgacctg caggcatgca agcttggcgt aatcagctgg acgtctttga cgaactcaag
     5761 gctatcgtcg atggccacct cgaccagtgc ggcggggtac tgctgcgtgg tttcgacgtg
     5821 ggcggagtca cggcctttcg cgaattcgcc caggccttcg gccacccgct gctcaactac
     5881 gagttcggct cgacgccgcg cagcaatgtt acccaagggg tctacacctc cacggagtac
     5941 cccgcgcacc agagcatccc gctgcacaac gagcaggcct acagcctcga ctggccgatg
     6001 cggatttggt tctacagtgt gatcgccgct cagactggcg gtgaaacgcc aattgccgac
     6061 agccgtgaag tgttccggcg catgccggcc gacatacgcg agcgctttgc cagcaagcgc
     6121 ctgatgtatg tgcgcaacta cggcaatggc cttgatgtga gttgggagca ggtgttcaac
     6181 accgaggacc gcagcgtagt ggaggcctat tgccgcgcgc accacatcga ctgggaatgg
     6241 aaggacgacg gtgagctgcg cacgcgccag gtctgccagg ccgtggccac gcacccctgg
     6301 acgggggatc aggtatggtt caatcaggcc cacctgttcc atgtctccaa cctaccggcc
     6361 gaggtgcgcg aaagcctgct ggaaatcgtc gacgaggaag acttgccgcg caacgtctac
     6421 tacggcgacg gcacaccgat cgagaacgaa gtgctcgaac gcattcgcgc tgtgctggac
     6481 gattgcgcga tcagtttccc ctggcaggaa aacgacgtgc tgatgctcga caacatgctc
     6541 gcggcgcacg cccgggcacc gttcaccggg ccacgcaaag tggtcgtggc catggctcag
     6601 gggcaccgcg aagcggcgct ttgaggccac tgtggcgagt gcttgcggct tgccgcaggc
     6661 gctcgccagc gcagcgtccg ggcatggcgc tgccgcgtta aattcgccct ccctccgctc
     6721 gttctactgg aaacgctttg gccgcacggc cgagctgcag atcagtaagg accccgagcc
     6781 gtgcgagctc atcgatgcat
//
