LOCUS       sequence-212715-        6793 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 6793)
  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 6793)
  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..6793
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..24,25..31)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    39..55
                     /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     44..66
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     63..79
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     63..79
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(2433..3227)
                     /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(2544..2563)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3154..3173
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(4408..4427)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4527..4549
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4587..4605)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4746..4850
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4851..4919,4920..5711)
                     /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     complement(5069..5088)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5882..6470
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6371..6390
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6624..6641
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6758..6779
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
       61 cacaggaaac agctatgacg gcatcgatct cgatgcccac ccgcgcgccg cgcccgctgg
      121 gcgccaccgg ccgggagcga ttggtcggca ggctgagcac cggttgctcc ccgccgaggg
      181 tgtccttcca gtaagccagc tgccgcgcct gctcacccgc ttccatccag ttgcgctgcc
      241 agatggcata gtcggcgtat tgcaccggca gggccggcag attcggtgcc tgcccctggc
      301 tgaggcgttc gtagcactgt accaaatcgt tgatcaagat cggcatcgac cagccgtcag
      361 cggcaatatg gtgcacggtc agcaccagca cgtggtcgtc ttcgccaagc ggcagcaggt
      421 tcagccgcaa cagcggccct tgttcaagat cgaacggctt ggcgatgcac gcctcgacgt
      481 gctcgcgcaa ggcgtcgctg gtcgcgctca tgccgatgag cggctcgatg agcaactcga
      541 acggcatctg cgggtggatg atctgcacca cgcggtcgct gtcttgtcgc aacgtggtgc
      601 gcagcacttc gtggcgctgg atcagcgcct ggcaggcacc gcgcaagccc tcgatatcca
      661 gccgcccctg cagctgcagc gccgcaccga tgttgtaggc tccgctggcg gggtccagtt
      721 gccagagaaa ccactgccgc tcctgggcgt aggacaccgg ggtcgggtcg gccgggtcgc
      781 gggcgaagat gcgcgtcacc ccgtagaggt tgacaccctg ctgcttgagc aaggcggcca
      841 gggccttgcg ctcccgggac gacagtgact ccacggaatc gagcaatgct tgcacttgaa
      901 catctcctac cagggcaccg gtctttgcct gcggaacgtg ccagcccgcg acaggctcat
      961 gatgctgccg aacaaatgaa agagccggac ggctgccggc tctgggtgta ctgaagttga
     1021 cgagccgggc ccggtcatac cgggcccgca gagcgctact gggtcaactc gtcgatctcc
     1081 tctgcagtaa gcatttcaag cctcagtcaa ttcattcatc agttgttcga taccggcgag
     1141 gccttcttcg gtcagggcct ggcccgcgcc ttccagcgcc tgggcaaagg cccgcagaac
     1201 cggtgtttcg aagatgagac ggatcggcat atcgatgccc agttggctgt tgatacgtga
     1261 gagggcctgg gccgccagca gcgagtgacc acccagttca aagaagttgt cgtccagccc
     1321 gacctgctcg atgcgcagga catcggccca gatcgctgcg accttgcatt cggtatcgct
     1381 gtacggcggc acatagcgca cctgaagctg ggccagatcg ggttgcggca gcgccttgcg
     1441 gtccagcttg ccgttcgggc tcaggggcag ctcgggcaac catatgaggt gggcaggcac
     1501 catgtagtcc ggcaggctgc tgcgaaggtc ggtgcgcaac tgcgccagaa gctcctgttg
     1561 cgtttgcacg ctggcctggg cgacgtctgc caccgcaggc accaggtagc caaccagttg
     1621 cttgcccagc ggcccatcga cgtcgatgac cagggcatca cgtacgctgg cgtgctccag
     1681 caggcgcgcg ccgacttctc ccagctcgat gcggaagccg cgaatcttca cctgatggtc
     1741 gatgcgcccg gcatattcga tctgcccatc ggcgaggtag cgcgccaggt cgccggagcg
     1801 atagaggcgg ctgccggggg cgccgaacgg gtcggggatg aagcgctcag cggtcaggcc
     1861 agggcgctgg tggtagcccc tggcgagcag ttcgccgccg atcatcagtt cgccgaccgc
     1921 gccattgagc acagcaccgc cgctgttatc cagcaggtgg atgctgcgcc cggccagcac
     1981 ccggccgatc ggcatcaccg gtggcagcgc ctgcgccgtg gcacctaggt aaggcgcgca
     2041 gtctgattac gccaagcttg catgcctgca ggtcgactct agaggatccg ggaccaaagc
     2101 ggccatcgtg cctccccact cctgcagttc gggggcatgg atgcgcggat agccgctgct
     2161 ggtttcctgg atgccgacgg atttgcactg ccggtagaac tccgcgaggt cgtccagcct
     2221 caggcagcag ctgaaccaac tcgcgagggg atcgagcccg gggtgggcga agaactccag
     2281 catgagatcc ccgcgctgga ggatcatcca gccggcgtcc cggaaaacga ttccgaagcc
     2341 caacctttca tagaaggcgg cggtggaatc gaaatctcgt gatggcaggt tgggcgtcgc
     2401 ttggtcggtc atttcgaacc ccagagtccc gctcagaaga actcgtcaag aaggcgatag
     2461 aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc
     2521 cattcgccgc caagctcttc agcaatatca cgggtagcca acgctatgtc ctgatagcgg
     2581 tccgccacac ccagccggcc acagtcgatg aatccagaaa agcggccatt ttccaccatg
     2641 atattcggca agcaggcatc gccatgggtc acgacgagat cctcgccgtc gggcatgcgc
     2701 gccttgagcc tggcgaacag ttcggctggc gcgagcccct gatgctcttc gtccagatca
     2761 tcctgatcga caagaccggc ttccatccga gtacgtgctc gctcgatgcg atgtttcgct
     2821 tggtggtcga atgggcaggt agccggatca agcgtatgca gccgccgcat tgcatcagcc
     2881 atgatggata ctttctcggc aggagcaagg tgagatgaca ggagatcctg ccccggcact
     2941 tcgcccaata gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac agctgcgcaa
     3001 ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct cgtcctgcag ttcattcagg
     3061 gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga cagccggaac
     3121 acggcggcat cagagcagcc gattgtctgt tgtgcccagt catagccgaa tagcctctcc
     3181 acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt caatcatgcg aaacgatcct
     3241 catcctgtct cttgatcaga tcttgatccc ctgcgccatc agatccttgg cggcaagaaa
     3301 gccatccagt ttactttgca gggcttccca accttaccag agggcgcccc agctggcaat
     3361 tccggttcgc ttgctgtcca taaaaccgcc cagtctagct atcgccatgt aagcccactg
     3421 caagctacct gctttctctt tgcgcttgcg ttttcccttg tccagatagc ccagtagctg
     3481 acattcatcc ggggtcagca ccgtttctgc ggactggctt tctacgtgtt ccgcttcctt
     3541 tagcagccct tgcgccctga gtgcttgcgg cagcgtgaag cttatcgata cgtagccggc
     3601 tagcattttg gaaaagcggg caaaggtacg catccgctcg tcacagatca agcatttatg
     3661 gactaacgtc cagaatgacc agacgacagc acaggctgaa tagattaatc tagcgcggtt
     3721 tttttgcccg cctttcggag ccctccccta tgcccactcg tgagatccgc catccgctga
     3781 tccgccacaa gcttggcctg atgcgccgtg ccgatatcag caccaagaat tttcgcgaac
     3841 tcgcccagga agtcggcgcg ctcctgacgt atgaagccac ccaggacctg cccctcgaaa
     3901 cctacgagat cgacggctgg tgtggcaagg tctcggttga aaaaatcgcc ggcaagaaga
     3961 tcaccgtagt accgatcctg cgcgccggca tcggcatgct cgacggcgtg ctcagcctga
     4021 tcccgggggc caaggtcagc gccgtcggcg ttgcccgaaa cgaagaaacc ctcgaagccc
     4081 acacctatct ggaaaagctc gcgccggaca tcaaccagcg cctggccctg atcatcgacc
     4141 cgatgctggc caccggcggc tcgatggttg ccaccatcga cctgctgaaa aaggccggct
     4201 gcaaagagat tcgcgccatg gtgctggtcg ccgcaccaga aggcatcgag gtggtggaaa
     4261 aagcccaccc ggacgtgaag atctacaccg cctcgatcga ccagcgcctg aacgagcacg
     4321 gctacatcgt gccaggcctg ggtgatgccg gtgacaagat cttcggcacc aagcaaaagg
     4381 acgcctgacc atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     4441 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     4501 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     4561 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     4621 catgataata atggtttctt agagtaggcc cacccgcgag cgggtgttcc ttcttcactg
     4681 tcccttctag aacgttctag aagggacagt gaagaaggaa aggtggcact tttcggggaa
     4741 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     4801 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     4861 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     4921 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     4981 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     5041 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     5101 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     5161 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     5221 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     5281 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     5341 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     5401 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     5461 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     5521 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     5581 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     5641 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     5701 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     5761 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     5821 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     5881 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     5941 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     6001 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     6061 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     6121 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     6181 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     6241 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     6301 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     6361 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     6421 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     6481 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     6541 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     6601 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     6661 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     6721 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
     6781 aggcacccca ggc
//
