LOCUS       sequence-451911-        4119 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 4119)
  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 4119)
  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: pGNW2
FEATURES             Location/Qualifiers
     source          1..4119
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(65..159)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1155..1949
                     /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(1209..1228)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1819..1838
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    2224..2245
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2260..2265,2266..2283,2284..2290)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2298..2314
                     /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     2303..2325
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2322..2338
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2322..2338
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2334..2645
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPDYPVIPRSLHACRSTLEDPRVPSSNSDYPVIPILAVVLQ
                     RRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWGSIPSRVGSAAA"
     misc_feature    2372..2428
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(2450..2467)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2450..2466)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2459..2481)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(2898..3269)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            3302..3411
                     /note="incP origin of transfer"
     rep_origin      3571..3959
                     /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 taaatggcga tagctagact gggcggtttt atggacagca agcgaaccgg
     1021 aattgccagc tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta aactggatgg
     1081 ctttcttgcc gccaaggatc tgatggcgca ggggatcaag atctgatcaa gagacaggat
     1141 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     1201 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     1261 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     1321 ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     1381 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     1441 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     1501 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     1561 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     1621 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     1681 cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     1741 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     1801 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     1861 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     1921 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
     1981 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
     2041 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     2101 gctggagttc ttcgcccacc ccccaatacg caaaccgcct ctccccgcgc gttggccgat
     2161 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     2221 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
     2281 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
     2341 tgattacgcc ggattaccct gttatcccta gaagcttgca tgcctgcagg tcgactctag
     2401 aggatccccg ggtaccgagc tcgaattcag attaccctgt tatccctata ctggccgtcg
     2461 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     2521 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     2581 agttgcgcag cctgaatggc gaatggggct cgatcccctc gcgagttggt tcagctgctg
     2641 cctgaggctg gacgacctcg cggagttcta ccggcagtgc aaatccgtcg gcatccagga
     2701 aaccagcagc ggctatccgc gcatccatgc ccccgaactg caggagtggg gaggcacgat
     2761 ggccgctttg gtcggtcttg ccttgctcgt cggtgatgta cttcaccagc tccgcgaagt
     2821 cgctcttctt gatggagcgc atggggacgt gcttggcaat cacgcgcacc ccccggccgt
     2881 tttagcggct aaaaaagtca tggctctgcc ctcgggcgga ccacgcccat catgaccttg
     2941 ccaagctcgt cctgcttctc ttcgatcttc gccagcaggg cgaggatcgt ggcatcaccg
     3001 aaccgcgccg tgcgcgggtc gtcggtgagc cagagtttca gcaggccgcc caggcggccc
     3061 aggtcgccat tgatgcgggc cagctcgcgg acgtgctcat agtccacgac gcccgtgatt
     3121 ttgtagccct ggccgacggc cagcaggtag gccgacaggc tcatgccggc cgccgccgcc
     3181 ttttcctcaa tcgctcttcg ttcgtctgga aggcagtaca ccttgatagg tgggctgccc
     3241 ttcctggttg gcttggtttc atcagccatc cgcttgccct catctgttac gccggcggta
     3301 gccggccagc ctcgcagagc aggattcccg ttgagcaccg ccaggtgcga ataagggaca
     3361 gtgaagaagg aacacccgct cgcgggtggg cctacttcac ctatcctgcc cggctgacgc
     3421 cgttggatac accaaggaaa gtctacacga accctttggc aaaatcctgt atatcgtgcg
     3481 aaaaaggatg gatataccga aaaaatcgct ataatgaccc cgaagcaggg ttatgcagcg
     3541 gaaaacggcc acgatgcgtc cggcgtagag gatctgaaga tcagcagttc aacctgttga
     3601 tagtacgtac taagctctca tgtttcacgt actaagctct catgtttaac gtactaagct
     3661 ctcatgttta acgaactaaa ccctcatggc taacgtacta agctctcatg gctaacgtac
     3721 taagctctca tgtttcacgt actaagctct catgtttgaa caataaaatt aatataaatc
     3781 agcaacttaa atagcctcta aggttttaag ttttataaga aaaaaaagaa tatataaggc
     3841 ttttaaagct tttaaggttt aacggttgtg gacaacaagc cagggatgta acgcactgag
     3901 aagcccttag agcctctcaa agcaattttg agtgacacag gaacacttaa cggctgacat
     3961 gggaattagc ttcacgctgc cgcaagcact cagggcgcaa gggctgctaa aggaagcgga
     4021 acacgtagaa agccagtccg cagaaacggt gctgaccccg gatgaatgtc agctactggg
     4081 ctatctggac aagggaaaac gcaagcgcaa agagaaagc
//
