LOCUS       sequence-243567-        3798 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 3798)
  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 3798)
  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: pGNW6
FEATURES             Location/Qualifiers
     source          1..3798
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(65..159)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(1033..1566)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(1755..1760,1761..1777,1778..1783))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     protein_bind    1903..1924
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1939..1944,1945..1962,1963..1969)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1977..1993
                     /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     1982..2004
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2001..2017
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2001..2017
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2013..2324
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPDYPVIPRSLHACRSTLEDPRVPSSNSDYPVIPILAVVLQ
                     RRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWGSIPSRVGSAAA"
     misc_feature    2051..2107
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(2129..2146)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2129..2145)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2138..2160)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(2577..2948)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            2981..3090
                     /note="incP origin of transfer"
     rep_origin      3250..3638
                     /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 taagaaccta atttaccgaa caactccgcg gccgggaagc cgatctcggc
     1021 ttgaacgaat tgttaggtgg cggtacttgg gtcgatatca aagtgcatca cttcttcccg
     1081 tatgcccaac tttgtataga gagccactgc gggatcgtca ccgtaatctg cttgcacgta
     1141 gatcacataa gcaccaagcg cgttggcctc atgcttgagg agattgatga gcgcggtggc
     1201 aatgccctgc ctccggtgct ctccggagac tgcgagatca tagatataga tctcactacg
     1261 cggctgctca aacttgggca gaacgtaagc cgcgagagcg ccaacaaccg cttcttggtc
     1321 gaaggcagca agcgcgatga atgtcttact acggagcaag ttcccgaggt aatcggagtc
     1381 cggctgatgt tgggagtagg tggctacgtc tccgaactca cgaccgaaaa gatcaagagc
     1441 agcccgcatg gatttgactt ggtcagggcc gagcctacat gtgcgaatga tgcccatact
     1501 tgagccacct aactttgttt tagggcgact gccctgctgc gtaacatcgt tgctgctgcg
     1561 taacatcgtt gctgctccat aacatcaaac atcgacccac ggcgtaacgc gcttgctgct
     1621 tggatgcccg aggcataggc tgtacaaaaa aacagtcata acaagccatg aaaaccgcca
     1681 ctgcgccgtt accaccgctg cgttcggtca aggttctgga ccagttgcgt gagcgcatac
     1741 gctacttgca ttacagttta cgaaccgaac aggcttatgt caattgtcct tcgcccaccc
     1801 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     1861 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
     1921 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     1981 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgccg gattaccctg
     2041 ttatccctag aagcttgcat gcctgcaggt cgactctaga ggatccccgg gtaccgagct
     2101 cgaattcaga ttaccctgtt atccctatac tggccgtcgt tttacaacgt cgtgactggg
     2161 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     2221 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     2281 aatggggctc gatcccctcg cgagttggtt cagctgctgc ctgaggctgg acgacctcgc
     2341 ggagttctac cggcagtgca aatccgtcgg catccaggaa accagcagcg gctatccgcg
     2401 catccatgcc cccgaactgc aggagtgggg aggcacgatg gccgctttgg tcggtcttgc
     2461 cttgctcgtc ggtgatgtac ttcaccagct ccgcgaagtc gctcttcttg atggagcgca
     2521 tggggacgtg cttggcaatc acgcgcaccc cccggccgtt ttagcggcta aaaaagtcat
     2581 ggctctgccc tcgggcggac cacgcccatc atgaccttgc caagctcgtc ctgcttctct
     2641 tcgatcttcg ccagcagggc gaggatcgtg gcatcaccga accgcgccgt gcgcgggtcg
     2701 tcggtgagcc agagtttcag caggccgccc aggcggccca ggtcgccatt gatgcgggcc
     2761 agctcgcgga cgtgctcata gtccacgacg cccgtgattt tgtagccctg gccgacggcc
     2821 agcaggtagg ccgacaggct catgccggcc gccgccgcct tttcctcaat cgctcttcgt
     2881 tcgtctggaa ggcagtacac cttgataggt gggctgccct tcctggttgg cttggtttca
     2941 tcagccatcc gcttgccctc atctgttacg ccggcggtag ccggccagcc tcgcagagca
     3001 ggattcccgt tgagcaccgc caggtgcgaa taagggacag tgaagaagga acacccgctc
     3061 gcgggtgggc ctacttcacc tatcctgccc ggctgacgcc gttggataca ccaaggaaag
     3121 tctacacgaa ccctttggca aaatcctgta tatcgtgcga aaaaggatgg atataccgaa
     3181 aaaatcgcta taatgacccc gaagcagggt tatgcagcgg aaaacggcca cgatgcgtcc
     3241 ggcgtagagg atctgaagat cagcagttca acctgttgat agtacgtact aagctctcat
     3301 gtttcacgta ctaagctctc atgtttaacg tactaagctc tcatgtttaa cgaactaaac
     3361 cctcatggct aacgtactaa gctctcatgg ctaacgtact aagctctcat gtttcacgta
     3421 ctaagctctc atgtttgaac aataaaatta atataaatca gcaacttaaa tagcctctaa
     3481 ggttttaagt tttataagaa aaaaaagaat atataaggct tttaaagctt ttaaggttta
     3541 acggttgtgg acaacaagcc agggatgtaa cgcactgaga agcccttaga gcctctcaaa
     3601 gcaattttga gtgacacagg aacacttaac ggctgacatg ggaattagct tcacgctgcc
     3661 gcaagcactc agggcgcaag ggctgctaaa ggaagcggaa cacgtagaaa gccagtccgc
     3721 agaaacggtg ctgaccccgg atgaatgtca gctactgggc tatctggaca agggaaaacg
     3781 caagcgcaaa gagaaagc
//
