LOCUS       sequence-335415-        6013 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integration vector for deleting pvdIJD locus in P. putida.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6013)
  AUTHORS   Cook TB, Jacobson TB, Venkataraman MV, Hofstetter H, Amador-Noguez 
            D, Thomas MG, Pfleger BF
  TITLE     Stepwise genetic engineering of Pseudomonas putida enables robust 
            heterologous production of prodigiosin and glidobactin A.
  JOURNAL   Metab Eng. 2021 Jun 24;67:112-124. doi: 10.1016/j.ymben.2021.06.004.
  PUBMED    34175462
REFERENCE   2  (bases 1 to 6013)
  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: "Metab 
            Eng."; date: "2021-06-24"; volume: "67:112-124. doi"; pages: " 
            10.1016/j.ymben.2021.06.004"
COMMENT     Sequence Label: pNVL-pvdIJD
FEATURES             Location/Qualifiers
     source          1..6013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             299..1093
                     /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(353..372)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     963..982
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3640..3662)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3651..3667
                     /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(3675..3681,3682..3699,3700..3705))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3720..3741
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3885..3902)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4019..4564)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(4099..4118)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4640..4659
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(join(4881..5672,5673..5741))
                     /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     5504..5523
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5742..5846)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5987..6005
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgagatg aatgtcagct actgggctat
       61 ctggacaagg gaaaacgcaa gcgcaaagag aaagcaggta gcttgcagtg ggcttacatg
      121 gcgatagcta gactgggcgg ttttatggac agcaagcgaa ccggaattgc cagctggggc
      181 gccctctggt aaggttggga agccctgcaa agtaaactgg atggctttct tgccgccaag
      241 gatctgatgg cgcaggggat caagatctga tcaagagaca ggatgaggat cgtttcgcat
      301 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
      361 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
      421 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
      481 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
      541 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
      601 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
      661 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
      721 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
      781 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
      841 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
      901 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
      961 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     1021 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     1081 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     1141 tcatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     1201 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     1261 caccccgggc tcgatcccct cgcgagttgg ttcagctgct gcctgaggct ggacgacctc
     1321 gcggagttct accggcagtg caaatccgtc ggcatccagg aaaccagcag cggctatccg
     1381 cgcatccatg cccccgaact gcaggagtgg ggaggcacga tggccgcttt ggtcccggat
     1441 cctctagagt cgacctgcag gcatgcaagc ttggcgtaat cagctggacg tctttgacga
     1501 actcaaggct atcgtcgatg gccacctcga ccagtgcggc ggggtactgc tgcgtggttt
     1561 cgacgtgggc ggagtcacgg cctttcgcga attcgcccag gccttcggcc acccgctgct
     1621 caactacgag ttcggctcga cgccgcgcag caatgttacc caaggggtct acacctccac
     1681 ggagtacccc gcgcaccaga gcatcccgct gcacaacgag caggcctaca gcctcgactg
     1741 gccgatgcgg atttggttct acagtgtgat cgccgctcag actggcggtg aaacgccaat
     1801 tgccgacagc cgtgaagtgt tccggcgcat gccggccgac atacgcgagc gctttgccag
     1861 caagcgcctg atgtatgtgc gcaactacgg caatggcctt gatgtgagtt gggagcaggt
     1921 gttcaacacc gaggaccgca gcgtagtgga ggcctattgc cgcgcgcacc acatcgactg
     1981 ggaatggaag gacgacggtg agctgcgcac gcgccaggtc tgccaggccg tggccacgca
     2041 cccctggacg ggggatcagg tatggttcaa tcaggcccac ctgttccatg tctccaacct
     2101 accggccgag gtgcgcgaaa gcctgctgga aatcgtcgac gaggaagact tgccgcgcaa
     2161 cgtctactac ggcgacggca caccgatcga gaacgaagtg ctcgaacgca ttcgcgctgt
     2221 gctggacgat tgcgcgatca gtttcccctg gcaggaaaac gacgtgctga tgctcgacaa
     2281 catgctcgcg gcgcacgccc gggcaccgtt caccgggcca cgcaaagtgg tcgtggccat
     2341 ggctcagggg caccgcgaag cggcgctttg aggccactgt ggcgagtgct tgcggcttgc
     2401 cgcaggcgct cgccagcgca gcgtccgggc atggcgctgc cgcgttaaat tcgccctccc
     2461 tccgctcgtt ctactggaaa cgctttggcc gcacggccga gctgcagatc agtaaggacc
     2521 ccgagcatgg acatcgatcc gactcagggt gggacgacgt acaagcgctg ctcgaaccca
     2581 acctcacgca cgcctgcttc attgaacttt catgagcttt cggggccgcc agcgcggccc
     2641 ctctggagaa tcgaacgatg tccctgaacc ctgacctggc ggcctacctg caactggtgg
     2701 aggccggacg cagcgccggc aaggtgctgc ccatgcatgc cctggcggcg gacgaggcgc
     2761 gccgccagtt cgaagagtcc tcggcgctga ttgcgggcaa ggccgacgag ccggactgca
     2821 tcagcgacct ttcgttgacc acccgcgatg gccatacctt gccggtgcgc ttgtaccgcc
     2881 cgccgcaggc cgaccctgca ctggccggcg cggcgctgct gtacctgcat ggcggcggct
     2941 acgtggtggg cagcctcgat tcgcatgaca cgctgtgctg gaacctggcg caggatgccg
     3001 gggtgccggt gattgccgtg ggctaccggc tggcgccgca gtggcgtttc ccgacagcca
     3061 gtgacgatgc cctggatgcc tggcgctggt tggtcgagca ggccgaggcg ctgggcatcg
     3121 atgcgcagcg actggcggtg gtgggcgaca gtgtcggtgg tagcctggct accattctgg
     3181 ccaaccagtt ggctgcgcag cgcgagctgc cggcaccgcg cctgcaggta atgatctacc
     3241 cggtgaccga tgccagttgc cggcggcctt cggtgcagcg ctatggcagc ggttacctgc
     3301 ttgaagcgca gaccctggag tggttctacc agcaatacgc cacggtgccg gcagaccgtc
     3361 acgatccgcg tttttcaccg ttgctgggca gcgtcgccag caacagtgca cctgcactga
     3421 tgctgattgc cgagtgcgac ccgctgcatg accagggtgt cgcttatgca cggcacttgg
     3481 agcaagccgg ggtggcggtg cagcttgcgg tgatcccggg agtgacccat gacttcatgc
     3541 gtatgggttc gatcatcgag gaggccgatg aaggcctggc gatggtggtc gaggcactgc
     3601 agcagcatct gtaagggagc ctttcaggca tagctgtttc ctgtgtgaaa ttgttatccg
     3661 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     3721 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3781 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3841 gggcgccgtc agcagaatat gtgatacagg atatattccg cttcctcgct cactgactcg
     3901 ctacgctcgg tcgttcgact gcggcgagcg gaaatggctt acgaacgggg cggagatttc
     3961 ctggaagatg ccaggaagat acttaacagg gaagtgagag ggccgcggca aagccgtttt
     4021 tccataggct ccgcccccct gacaagcatc acgaaatctg acgctcaaat cagtggtggc
     4081 gaaacccgac aggactataa agataccagg cgtttccccc tggcggctcc ctcgtgcgct
     4141 ctcctgttcc tgcctttcgg tttaccggtg tcattccgct gttatggccg cgtttgtctc
     4201 attccacgcc tgacactcag ttccgggtag gcagttcgct ccaagctgga ctgtatgcac
     4261 gaaccccccg ttcagtccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4321 ccggaaagac atgcaaaagc accactggca gcagccactg gtaattgatt tagaggagtt
     4381 agtcttgaag tcatgcgccg gttaaggcta aactgaaagg acaagttttg gtgactgcgc
     4441 tcctccaagc cagttacctc ggttcaaaga gttggtagct cagagaacct tcgaaaaacc
     4501 gccctgcaag gcggtttttt cgttttcaga gcaagagatt acgcgcagac caaaacgatc
     4561 tcaagaagat catcttatta atcagataaa atatttctag gctcatgagc ccgaagtggc
     4621 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     4681 cgccggtgat gccggccacg atgcgtccgg cgtagaggat ctgctcatgt ttgacagctt
     4741 atcgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     4801 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     4861 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     4921 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     4981 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     5041 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     5101 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     5161 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     5221 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     5281 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     5341 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     5401 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     5461 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     5521 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     5581 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     5641 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     5701 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     5761 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     5821 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tttccttctt
     5881 cactgtccct tctagaacgt tctagaaggg acagtgaaga aggaacaccc gctcgcgggt
     5941 gggcctactc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac
     6001 gaggcccttt cgt
//
