LOCUS       sequence-266662-        4831 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  P. putida KT2440 rescue construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4831)
  AUTHORS   Graf N, Altenbuchner J
  TITLE     Development of a method for markerless gene deletion in Pseudomonas 
            putida.
  JOURNAL   Appl Environ Microbiol. 2011 Aug;77(15):5549-52. doi: 
            10.1128/AEM.05055-11. Epub 2011 Jun 10.
  PUBMED    21666018
REFERENCE   2  (bases 1 to 4831)
  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"; doi: 
            "10.1128/AEM.05055-11"; journalName: "Appl Environ Microbiol"; date:
            "2011-08"; volume: "77"; issue: "15"; pages: "5549-52"
COMMENT     Sequence Label: pJOE6261.2
FEATURES             Location/Qualifiers
     source          1..4831
                     /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(2538..2554)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2538..2554)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2551..2573)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2562..2578
                     /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(2586..2592,2593..2610,2611..2616))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2631..2652
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2769..2786)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2940..3528)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3020..3039)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3699..4490,4491..4559))
                     /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     4322..4341
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4560..4664)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4805..4823
                     /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 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     2581 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     2641 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     2701 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     2761 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     2821 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     2881 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     2941 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     3001 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     3061 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     3121 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     3181 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     3241 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     3301 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     3361 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     3421 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     3481 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     3541 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     3601 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     3661 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     3721 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     3781 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     3841 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     3901 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     3961 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     4021 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     4081 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     4141 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     4201 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     4261 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     4321 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     4381 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     4441 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     4501 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     4561 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     4621 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     4681 gtgccacctt tccttcttca ctgtcccttc tagaacgttc tagaagggac agtgaagaag
     4741 gaacacccgc tcgcgggtgg gcctactcta agaaaccatt attatcatga cattaaccta
     4801 taaaaatagg cgtatcacga ggccctttcg t
//
