LOCUS       sequence-232807-        3982 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  suicide vector for integration into Pseudomonas genome after 
            insertion of homologous regions at the multiple cloning site; 
            successful integration causes green fluorescence in cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3982)
  AUTHORS   Wirth NT, Kozaeva E, Nikel PI
  TITLE     Accelerated genome engineering of Pseudomonas putida by 
            I-SceI-mediated recombination and CRISPR-Cas9 counterselection.
  JOURNAL   Microb Biotechnol. 2019 Mar 12. doi: 10.1111/1751-7915.13396.
  PUBMED    30861315
REFERENCE   2  (bases 1 to 3982)
  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: "Microb 
            Biotechnol. 2019 Mar 12. doi: 10.1111/1751-7915.13396."
COMMENT     Sequence Label: pGNW4
FEATURES             Location/Qualifiers
     source          1..3982
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(65..159)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1171..1962
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     protein_bind    2087..2108
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2123..2128,2129..2146,2147..2153)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2161..2177
                     /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     2166..2188
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2185..2201
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2185..2201
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2197..2508
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPDYPVIPRSLHACRSTLEDPRVPSSNSDYPVIPILAVVLQ
                     RRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWGSIPSRVGSAAA"
     misc_feature    2235..2291
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(2313..2330)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2313..2329)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2322..2344)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(2761..3132)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            3165..3274
                     /note="incP origin of transfer"
     rep_origin      3434..3822
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
ORIGIN
        1 aggtagctgt aacatcagag attttgagac acaaatttaa atcgtaatta ttggggaccc
       61 ctggattctc accaataaaa aacgcccggc ggcaaccgag cgttctgaac aaatccagat
      121 ggagttctga ggtcattact ggatctatca acaggagtcc aagactagtc atttatttgt
      181 agagttcatc catgccgtgc gtgatacctg cggcagtaac gaactccagc agcaccatgt
      241 ggtcgcgctt ttcgttcggg tctttggaca gtttagactg ggtggacagg tagtggttat
      301 ccggcagcag aaccggacca tcaccgatcg gagtgttctg ctggtagtgg tccgccagct
      361 gtacgctacc gtcttcaacg ttatggcgaa ttttgaagtt agctttgata ccgttcttct
      421 gtttgtctgc ggtgatgtaa acgttatggg agttgaagtt atattccagt ttgtggccca
      481 ggatgttgcc gtcctctttg aaatcaatgc ctttcagttc aatacggttc accagagtat
      541 caccttcaaa tttaacctct gcacgggttt tgtaggtgcc atcgtctttg aaagaaatgg
      601 tgcgctcctg tacataacct tccggcattg cagatttgaa gaaatcatgc tgcttcatgt
      661 gatccgggta acgagaaaaa cactgaacac cataggtcag ggtagtcacc agagtcggcc
      721 atggaaccgg cagtttaccg gtagtgcaga tgaatttcag ggtcagttta ccgttggttg
      781 catcaccttc accttcacca cgaacagaga atttgtggcc gttaacatca ccatccagtt
      841 caaccaggat cggaacaaca ccggtgaaca gttcttcacc tttacgcata tgtttttcct
      901 cctaaacatg cgtcgcggcc taggtcgtgc aattatacct ggccgcgaga gccttgtcaa
      961 tgggcttaat taagaacctt gaccgaacgc agcggtggta acggcgcagt ggcggttttc
     1021 atggcttgtt atgactgttt ttttggggta cagtctatgc ctcgggcatc caagcagcaa
     1081 gcgcgttacg ccgtgggtcg atgtttgatg ttatggagca gcaacgatgt tacgcagcag
     1141 ggcagtcgcc ctaaaacaaa gttaaacatc atgagggaag cggtgatcgc cgaagtatcg
     1201 actcaactat cagaggtagt tggcgtcatc gagcgccatc tcgaaccgac gttgctggcc
     1261 gtacatttgt acggctccgc agtggatggc ggcctgaagc cacacagtga tattgatttg
     1321 ctggttacgg tgaccgtaag gcttgatgaa acaacgcggc gagctttgat caacgacctt
     1381 ttggaaactt cggcttcccc tggagagagc gagattctcc gcgctgtaga agtcaccatt
     1441 gttgtgcacg acgacatcat tccgtggcgt tatccagcta agcgcgaact gcaatttgga
     1501 gaatggcagc gcaatgacat tcttgcaggt atcttcgagc cagccacgat cgacattgat
     1561 ctggctatct tgctgacaaa agcaagagaa catagcgttg ccttggtagg tccagcggcg
     1621 gaggaactct ttgatccggt tcctgaacag gatctatttg aggcgctaaa tgaaacctta
     1681 acgctatgga actcgccgcc cgactgggct ggcgatgagc gaaatgtagt gcttacgttg
     1741 tcccgcattt ggtacagcgc agtaaccggc aaaatcgcgc cgaaggatgt cgctgccgac
     1801 tgggcaatgg agcgcctgcc ggcccagtat cagcccgtca tacttgaagc tagacaggct
     1861 tatcttggac aagaagaaga tcgcttggcc tcgcgcgcag atcagttgga agaatttgtc
     1921 cactacgtga aaggcgagat caccaaggta gtcggcaaat aagacaattg tccttcgccc
     1981 accccccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     2041 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
     2101 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
     2161 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccggattac
     2221 cctgttatcc ctagaagctt gcatgcctgc aggtcgactc tagaggatcc ccgggtaccg
     2281 agctcgaatt cagattaccc tgttatccct atactggccg tcgttttaca acgtcgtgac
     2341 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     2401 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     2461 ggcgaatggg gctcgatccc ctcgcgagtt ggttcagctg ctgcctgagg ctggacgacc
     2521 tcgcggagtt ctaccggcag tgcaaatccg tcggcatcca ggaaaccagc agcggctatc
     2581 cgcgcatcca tgcccccgaa ctgcaggagt ggggaggcac gatggccgct ttggtcggtc
     2641 ttgccttgct cgtcggtgat gtacttcacc agctccgcga agtcgctctt cttgatggag
     2701 cgcatgggga cgtgcttggc aatcacgcgc accccccggc cgttttagcg gctaaaaaag
     2761 tcatggctct gccctcgggc ggaccacgcc catcatgacc ttgccaagct cgtcctgctt
     2821 ctcttcgatc ttcgccagca gggcgaggat cgtggcatca ccgaaccgcg ccgtgcgcgg
     2881 gtcgtcggtg agccagagtt tcagcaggcc gcccaggcgg cccaggtcgc cattgatgcg
     2941 ggccagctcg cggacgtgct catagtccac gacgcccgtg attttgtagc cctggccgac
     3001 ggccagcagg taggccgaca ggctcatgcc ggccgccgcc gccttttcct caatcgctct
     3061 tcgttcgtct ggaaggcagt acaccttgat aggtgggctg cccttcctgg ttggcttggt
     3121 ttcatcagcc atccgcttgc cctcatctgt tacgccggcg gtagccggcc agcctcgcag
     3181 agcaggattc ccgttgagca ccgccaggtg cgaataaggg acagtgaaga aggaacaccc
     3241 gctcgcgggt gggcctactt cacctatcct gcccggctga cgccgttgga tacaccaagg
     3301 aaagtctaca cgaacccttt ggcaaaatcc tgtatatcgt gcgaaaaagg atggatatac
     3361 cgaaaaaatc gctataatga ccccgaagca gggttatgca gcggaaaacg gccacgatgc
     3421 gtccggcgta gaggatctga agatcagcag ttcaacctgt tgatagtacg tactaagctc
     3481 tcatgtttca cgtactaagc tctcatgttt aacgtactaa gctctcatgt ttaacgaact
     3541 aaaccctcat ggctaacgta ctaagctctc atggctaacg tactaagctc tcatgtttca
     3601 cgtactaagc tctcatgttt gaacaataaa attaatataa atcagcaact taaatagcct
     3661 ctaaggtttt aagttttata agaaaaaaaa gaatatataa ggcttttaaa gcttttaagg
     3721 tttaacggtt gtggacaaca agccagggat gtaacgcact gagaagccct tagagcctct
     3781 caaagcaatt ttgagtgaca caggaacact taacggctga catgggaatt agcttcacgc
     3841 tgccgcaagc actcagggcg caagggctgc taaaggaagc ggaacacgta gaaagccagt
     3901 ccgcagaaac ggtgctgacc ccggatgaat gtcagctact gggctatctg gacaagggaa
     3961 aacgcaagcg caaagagaaa gc
//
