LOCUS       sequence-207209-        6792 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Suicide plasmid with repair template for knockout in P. putida.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6792)
  AUTHORS   Cook TB, Rand JM, Nurani W, Courtney DK, Liu SA, Pfleger BF
  TITLE     Genetic tools for reliable gene expression and recombineering in 
            Pseudomonas putida.
  JOURNAL   J Ind Microbiol Biotechnol. 2018 Jan 3. pii: 
            10.1007/s10295-017-2001-5. doi: 10.1007/s10295-017-2001-5.
  PUBMED    29299733
REFERENCE   2  (bases 1 to 6792)
  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 Ind 
            Microbiol Biotechnol. 2018 Jan 3. pii: 10.1007/s10295-017-2001-5. 
            doi: 10.1007/s10295-017-2001-5."
COMMENT     Sequence Label: pJOE_pvdJ
FEATURES             Location/Qualifiers
     source          1..6792
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(30..52)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     152..171
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             1352..2146
                     /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(1406..1425)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2016..2035
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4500..4516)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4500..4516)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4513..4535)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4524..4540
                     /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)."
     promoter        complement(join(4548..4554,4555..4572,4573..4578))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4593..4614
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4731..4748)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4902..5490)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4982..5001)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5661..6452,6453..6521))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6284..6303
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6522..6626)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6766..6784
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
       61 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
      121 gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg
      181 caccatatgg tcaggcgtcc ttttgcttgg tgccgaagat cttgtcaccg gcatcaccca
      241 ggcctggcac gatgtagccg tgctcgttca ggcgctggtc gatcgaggcg gtgtagatct
      301 tcacgtccgg gtgggctttt tccaccacct cgatgccttc tggtgcggcg accagcacca
      361 tggcgcgaat ctctttgcag ccggcctttt tcagcaggtc gatggtggca accatcgagc
      421 cgccggtggc cagcatcggg tcgatgatca gggccaggcg ctggttgatg tccggcgcga
      481 gcttttccag ataggtgtgg gcttcgaggg tttcttcgtt tcgggcaacg ccgacggcgc
      541 tgaccttggc ccccgggatc aggctgagca cgccgtcgag catgccgatg ccggcgcgca
      601 ggatcggtac tacggtgatc ttcttgccgg cgattttttc aaccgagacc ttgccacacc
      661 agccgtcgat ctcgtaggtt tcgaggggca ggtcctgggt ggcttcatac gtcaggagcg
      721 cgccgacttc ctgggcgagt tcgcgaaaat tcttggtgct gatatcggca cggcgcatca
      781 ggccaagctt gtggcggatc agcggatggc ggatctcacg agtgggcata ggggagggct
      841 ccgaaaggcg ggcaaaaaaa ccgcgctaga ttaatctatt cagcctgtgc tgtcgtctgg
      901 tcattctgga cgttagtcca taaatgcttg atctgtgacg agcggatgcg tacctttgcc
      961 cgcttttcca aaatgctagc cggctacgta tcgataagct tcacgctgcc gcaagcactc
     1021 agggcgcaag ggctgctaaa ggaagcggaa cacgtagaaa gccagtccgc agaaacggtg
     1081 ctgaccccgg atgaatgtca gctactgggc tatctggaca agggaaaacg caagcgcaaa
     1141 gagaaagcag gtagcttgca gtgggcttac atggcgatag ctagactggg cggttttatg
     1201 gacagcaagc gaaccggaat tgccagctgg ggcgccctct ggtaaggttg ggaagccctg
     1261 caaagtaaac tggatggctt tcttgccgcc aaggatctga tggcgcaggg gatcaagatc
     1321 tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     1381 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     1441 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     1501 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct
     1561 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     1621 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     1681 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     1741 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     1801 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     1861 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     1921 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     1981 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     2041 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     2101 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     2161 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     2221 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     2281 cagcgcgggg atctcatgct ggagttcttc gcccaccccg ggctcgatcc cctcgcgagt
     2341 tggttcagct gctgcctgag gctggacgac ctcgcggagt tctaccggca gtgcaaatcc
     2401 gtcggcatcc aggaaaccag cagcggctat ccgcgcatcc atgcccccga actgcaggag
     2461 tggggaggca cgatggccgc tttggtcccg gatcctctag agtcgacctg caggcatgca
     2521 agcttggcgt aatcagactg cgcgccttac ctaggtgcca cggcgcaggc gctgccaccg
     2581 gtgatgccga tcggccgggt gctggccggg cgcagcatcc acctgctgga taacagcggc
     2641 ggtgctgtgc tcaatggcgc ggtcggcgaa ctgatgatcg gcggcgaact gctcgccagg
     2701 ggctaccacc agcgccctgg cctgaccgct gagcgcttca tccccgaccc gttcggcgcc
     2761 cccggcagcc gcctctatcg ctccggcgac ctggcgcgct acctcgccga tgggcagatc
     2821 gaatatgccg ggcgcatcga ccatcaggtg aagattcgcg gcttccgcat cgagctggga
     2881 gaagtcggcg cgcgcctgct ggagcacgcc agcgtacgtg atgccctggt catcgacgtc
     2941 gatgggccgc tgggcaagca actggttggc tacctggtgc ctgcggtggc agacgtcgcc
     3001 caggccagcg tgcaaacgca acaggagctt ctggcgcagt tgcgcaccga ccttcgcagc
     3061 agcctgccgg actacatggt gcctgcccac ctcatatggt tgcccgagct gcccctgagc
     3121 ccgaacggca agctggaccg caaggcgctg ccgcaacccg atctggccca gcttcaggtg
     3181 cgctatgtgc cgccgtacag cgataccgaa tgcaaggtcg cagcgatctg ggccgatgtc
     3241 ctgcgcatcg agcaggtcgg gctggacgac aacttctttg aactgggtgg tcactcgctg
     3301 ctggcggccc aggccctctc acgtatcaac agccaactgg gcatcgatat gccgatccgt
     3361 ctcatcttcg aaacaccggt tctgcgggcc tttgcccagg cgctggaagg cgcgggccag
     3421 gccctgaccg aagaaggcct cgccggtatc gaacaactga tgaatgaatt gactgaggct
     3481 tgaaatgctt actgcagagg agatcgacga gttgacccag tagcgctctg cgggcccggt
     3541 atgaccgggc ccggctcgtc aacttcagta cacccagagc cggcagccgt ccggctcttt
     3601 catttgttcg gcagcatcat gagcctgtcg cgggctggca cgttccgcag gcaaagaccg
     3661 gtgccctggt aggagatgtt caagtgcaag cattgctcga ttccgtggag tcactgtcgt
     3721 cccgggagcg caaggccctg gccgccttgc tcaagcagca gggtgtcaac ctctacgggg
     3781 tgacgcgcat cttcgcccgc gacccggccg acccgacccc ggtgtcctac gcccaggagc
     3841 ggcagtggtt tctctggcaa ctggaccccg ccagcggagc ctacaacatc ggtgcggcgc
     3901 tgcagctgca ggggcggctg gatatcgagg gcttgcgcgg tgcctgccag gcgctgatcc
     3961 agcgccacga agtgctgcgc accacgttgc gacaagacag cgaccgcgtg gtgcagatca
     4021 tccacccgca gatgccgttc gagttgctca tcgagccgct catcggcatg agcgcgacca
     4081 gcgacgcctt gcgcgagcac gtcgaggcgt gcatcgccaa gccgttcgat cttgaacaag
     4141 ggccgctgtt gcggctgaac ctgctgccgc ttggcgaaga cgaccacgtg ctggtgctga
     4201 ccgtgcacca tattgccgct gacggctggt cgatgccgat cttgatcaac gatttggtac
     4261 agtgctacga acgcctcagc caggggcagg caccgaatct gccggccctg ccggtgcaat
     4321 acgccgacta tgccatctgg cagcgcaact ggatggaagc gggtgagcag gcgcggcagc
     4381 tggcttactg gaaggacacc ctcggcgggg agcaaccggt gctcagcctg ccgaccaatc
     4441 gctcccggcc ggtggcgccc agcgggcgcg gcgcgcgggt gggcatcgag atcgatgccg
     4501 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     4561 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     4621 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4681 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     4741 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     4801 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4861 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4921 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4981 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5041 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     5101 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5161 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5221 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     5281 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     5341 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     5401 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     5461 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     5521 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     5581 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     5641 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     5701 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     5761 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     5821 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     5881 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     5941 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     6001 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     6061 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     6121 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6181 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6241 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     6301 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     6361 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     6421 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     6481 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     6541 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     6601 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctag
     6661 aagggacagt gaagaaggaa cacccgctcg cgggtgggcc tacttcacct atcctgcccg
     6721 gctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     6781 aggccctttc gt
//
