LOCUS       sequence-217897-        7748 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway compatible allelic exchange vector for Burkholderia sp..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7748)
  AUTHORS   Fazli M, Harrison JJ, Gambino M, Givskov M, Tolker-Nielsen T
  TITLE     In-Frame and Unmarked Gene Deletions in Burkholderia cenocepacia via
            an Allelic Exchange System Compatible with Gateway Technology.
  JOURNAL   Appl Environ Microbiol. 2015 Jun;81(11):3623-30. doi: 
            10.1128/AEM.03909-14. Epub 2015 Mar 20.
  PUBMED    25795676
REFERENCE   2  (bases 1 to 7748)
  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.03909-14"; journalName: "Appl Environ Microbiol"; date:
            "2015-06"; volume: "81"; issue: "11"; pages: "3623-30"
COMMENT     Sequence Label: pDONRPEX18Km-SceI-pheS
FEATURES             Location/Qualifiers
     source          1..7748
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(5..24)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6..24)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(29..45)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(118..134)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(131..153)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    142..158
                     /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(166..172,173..190,191..196))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    211..232
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(520..1108)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(600..619)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1479..2273)
                     /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(1590..1609)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2200..2219
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(2422..2427,2428..2444,2445..2450))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     promoter        complement(2468..2572)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2640..2658
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2696..2718)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2818..2837
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     oriT            4108..4217
                     /note="incP origin of transfer"
     terminator      complement(4660..4687)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4779..4865)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     5018..5035
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     primer_bind     5215..5231
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5288..5304
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5321..5552
                     /gene="mutant version of attP"
                     /label=attP1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(5948..6253)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(6050..6069)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(6573..7256)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     7171..7190
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(7257..7359)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(7504..7735)
                     /gene="mutant version of attP"
                     /label=attP2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 tatcccctat agtgagtcgt attacatggt catagctgtt tcctggcagc tctggcccgt
       61 gtctcaaaat ctctgatcta gaggatcccc gggtaccgag ctcgaattcg taatcatgtc
      121 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
      181 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
      241 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
      301 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
      361 cccgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
      421 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
      481 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
      541 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
      601 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
      661 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
      721 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
      781 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
      841 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
      901 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
      961 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     1021 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     1081 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     1141 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     1201 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     1261 gtaaacttgg tctgacaatc gatgcgaatt ggccatttgc ccatgggggt gggcgaagaa
     1321 ctccagcatg agatccccgc gctggaggat catccagccg gcgtcccgga aaacgattcc
     1381 gaagcccaac ctttcataga aggcggcggt ggaatcgaaa tctcgtgatg gcaggttggg
     1441 cgtcgcttgg tcggtcattt cgaaccccag agtcccgctc agaagaactc gtcaagaagg
     1501 cgatagaagg cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg
     1561 tcagcccatt cgccgccaag ctcttcagca atatcacggg tagccaacgc tatgtcctga
     1621 tagcggtccg ccacacccag ccggccacag tcgatgaatc cagaaaagcg gccattttcc
     1681 accatgatat tcggcaagca ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc
     1741 atgcgcgcct tgagcctggc gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc
     1801 agatcatcct gatcgacaag accggcttcc atccgagtac gtgctcgctc gatgcgatgt
     1861 ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca
     1921 tcagccatga tggatacttt ctcggcagga gcaaggtgag atgacaggag atcctgcccc
     1981 ggcacttcgc ccaatagcag ccagtccctt cccgcttcag tgacaacgtc gagcacagct
     2041 gcgcaaggaa cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc ctgcagttca
     2101 ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc
     2161 cggaacacgg cggcatcaga gcagccgatt gtctgttgtg cccagtcata gccgaatagc
     2221 ctctccaccc aagcggccgg agaacctgcg tgcaatccat cttgttcaat catgcgaaac
     2281 gatcctcatc ctgtctcttg atcagatcgt acaaaaaaac agtcataaca agccatgaaa
     2341 accgccactg cgccgttacc accgctgcgt tcggtcaagg ttctggacca gttgcgtgag
     2401 cgcatacgct acttgcatta cagtttacga accgaacagg cttatgtcaa ttcggttgaa
     2461 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     2521 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     2581 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa
     2641 ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct
     2701 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     2761 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg
     2821 catcagagca gattgtactg agagtgcacc ataatcggca ttttcttttg cgtttttatt
     2881 tgttaactgt taattgtcct tgttcaagga tgctgtcttt gacaacagat gttttcttgc
     2941 ctttgatgtt cagcaggaag ctaggcgcaa acgttggatc tttaggcgaa ctggcggagg
     3001 aaccggaggt cattttcgaa aaagagccgg aggtcctgga caccataccg gagcatggta
     3061 aggcgttcca acccggaacc gaagccgaag ccaatgtaac gctccgggtc gaggcccata
     3121 ttccgaatga ccgtggggtg gacctgaccg ctgccgctga tttccaacca cttaccagca
     3181 ttcttcccat gttcaaacat catatcaatt tccgcgctgg gttccgtgaa ggggaagtag
     3241 ctcgggcgga aacggaccaa gatgtcatct cgttcgaaaa atttcttgag aaagtcggtg
     3301 taggcaccct tgagatcggc aaacgacacg ttttcatcaa tccagagccc ctccacctga
     3361 ttaaacatgg ggctatgggt ggcgtccgaa tcgacccgat aggtccgacc gggagcaatc
     3421 accttaatcg ggggtcgatt catgcgggcg taccgcacct gcatgggcga ggtatgggtc
     3481 cggaggagga ggggccgccc atcggcgtcc ttaccatcca catagaaggt gtcctgcatg
     3541 gaccgtgcgg ggtggttttc gggcgagttg agcgccgtga aattatacca atccgtctca
     3601 atttccggtc catccgcgac atcgaagcca atgctacgga aaatctgctc cacacgttcc
     3661 caggttcgca tgacggggtg cagcgagccg gcgcccgccc cgcgaccggg gagggtcaca
     3721 tcaatggctt cagccgccag gcgctgattg agaagggcat ccgccaaggc ctgccgccgg
     3781 gcattcaagg ccgcctccac ctgctgcttg gccacgttaa tccgggcacc ctcggtcttc
     3841 cgggtttccg ggtccagctt acccagcccc ttcaagagtt ccgtgagggc accgctcttg
     3901 cccaggaagc gggccttttc attttccaag gtcgtaatgt cggccgcgcc ctcaaagctc
     3961 tgctgggcat ccgccacgat ctgatcgagg tccatatgct tgtcctttcg aagctcgatc
     4021 cgattgtcag acccggcatg atattccgaa aatcccaagc gagtcaacag ctagtgcttg
     4081 ccctcagtac tgttacgccg gcggtagccg gccagcctcg cagagcagga ttcccgttga
     4141 gcaccgccag gtgcgaataa gggacagtga agaaggaaca cccgctcgcg ggtgggccta
     4201 cttcacctat cctgcccggc tgacgccgtt ggatacacca aggaaagtct acacgaaccc
     4261 tttggcaaaa tcctgtatat cgtgcgaaaa aggatggata taccgaaaaa atcgctataa
     4321 tgaccccgaa gcagggttat gcagcggaaa agcgctgctt ccctgctgtt ttgtggaata
     4381 tctaccgact ggaaacaggc aaatgcaaga aattactgaa ctgaggggac aggcgagagg
     4441 cgatgccaaa gagctacacc gacgagctgg ccgagtgggt tgaatcccgc gcggccaaga
     4501 agcgccggcg tgatgaggct gcggttgcgt tcctggcggt gagggcggat gtcgatatgc
     4561 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc atatttgaat
     4621 gtatttagaa aaataaacaa aagagtttgt agaaacgcaa aaaggccatc cgtcaggatg
     4681 gccttctgct taatttgatg cctggcagtt tatggcgggc gtcctgcccg ccaccctccg
     4741 ggccgttgct tcgcaacgtt caaatccgct cccggcggat ttgtcctact caggagagcg
     4801 ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc ctttcgtttt
     4861 atttgatgcc tggcagttcc ctactctcgc atggggagac cccacactac catcggcgct
     4921 acggcgtttc acttctgagt tcggcatggg gtcaggtggg accaccgcgc tactgccgcc
     4981 aggcaaattc tgttttatca gaccgcttct gcgttctgat ttaatctgta tcaggctgaa
     5041 aatcttctct catccgccaa aacagccaag ctcgccattc gccattcagg ctgcgcaact
     5101 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
     5161 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
     5221 cgacggccag tgccaagctt tagggataac agggtaatag catggatgtt ttcccagtca
     5281 cgacgttgta aaacgacggc cagtcttaag ctcgggcccc aaataatgat tttattttga
     5341 ctgatagtga cctgttcgtt gcaacaaatt gatgagcaat gcttttttat aatgccaact
     5401 ttgtacaaaa aagctgaacg agaaacgtaa aatgatataa atatcaatat attaaattag
     5461 attttgcata aaaaacagac tacataatac tgtaaaacac aacatatcca gtcactatga
     5521 atcaactact tagatggtat tagtgacctg tagtcgaccg acagccttcc aaatgttctt
     5581 cgggtgatgc tgccaactta gtcgaccgac agccttccaa atgttcttct caaacggaat
     5641 cgtcgtatcc agcctactcg ctattgtcct caatgccgta ttaaatcata aaaagaaata
     5701 agaaaaagag gtgcgagcct cttttttgtg tgacaaaata aaaacatcta cctattcata
     5761 tacgctagtg tcatagtcct gaaaatcatc tgcatcaaga acaatttcac aactcttata
     5821 cttttctctt acaagtcgtt cggcttcatc tggattttca gcctctatac ttactaaacg
     5881 tgataaagtt tctgtaattt ctactgtatc gacctgcaga ctggctgtgt ataagggagc
     5941 ctgacattta tattccccag aacatcaggt taatggcgtt tttgatgtca ttttcgcggt
     6001 ggctgagatc agccacttct tccccgataa cggagaccgg cacactggcc atatcggtgg
     6061 tcatcatgcg ccagctttca tccccgatat gcaccaccgg gtaaagttca cgggagactt
     6121 tatctgacag cagacgtgca ctggccaggg ggatcaccat ccgtcgcccg ggcgtgtcaa
     6181 taatatcact ctgtacatcc acaaacagac gataacggct ctctctttta taggtgtaaa
     6241 ccttaaactg catttcacca gcccctgttc tcgtcagcaa aagagccgtt catttcaata
     6301 aaccgggcga cctcagccat cccttcctga ttttccgctt tccagcgttc ggcacgcaga
     6361 cgacgggctt cattctgcat ggttgtgctt accagaccgg agatattgac atcatatatg
     6421 ccttgagcaa ctgatagctg tcgctgtcaa ctgtcactgt aatacgctgc ttcatagcat
     6481 acctcttttt gacatacttc gggtatacat atcagtatat attcttatac cgcaaaaatc
     6541 agcgcgcaaa tacgcatact gttatctggc ttttagtaag ccggatccac gcggcgttta
     6601 cgccccccct gccactcatc gcagtactgt tgtaattcat taagcattct gccgacatgg
     6661 aagccatcac aaacggcatg atgaacctga atcgccagcg gcatcagcac cttgtcgcct
     6721 tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga agttgtccat attggccacg
     6781 tttaaatcaa aactggtgaa actcacccag ggattggctg agacgaaaaa catattctca
     6841 ataaaccctt tagggaaata ggccaggttt tcaccgtaac acgccacatc ttgcgaatat
     6901 atgtgtagaa actgccggaa atcgtcgtgg tattcactcc agagcgatga aaacgtttca
     6961 gtttgctcat ggaaaacggt gtaacaaggg tgaacactat cccatatcac cagctcaccg
     7021 tctttcattg ccatacggaa ttccggatga gcattcatca ggcgggcaag aatgtgaata
     7081 aaggccggat aaaacttgtg cttatttttc tttacggtct ttaaaaaggc cgtaatatcc
     7141 agctgaacgg tctggttata ggtacattga gcaactgact gaaatgcctc aaaatgttct
     7201 ttacgatgcc attgggatat atcaacggtg gtatatccag tgattttttt ctccatttta
     7261 gcttccttag ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag tgatcttatt
     7321 tcattatggt gaaagttgga acctcttacg tgccgatcaa cgtctcattt tcgccaaaag
     7381 ttggcccagg gcttcccggt atcaacaggg acaccaggat ttatttattc tgcgaagtga
     7441 tcttccgtca caggtattta ttcggcgcaa agtgcgtcgg gtgatgctgc caacttagtc
     7501 gactacaggt cactaatacc atctaagtag ttgattcata gtgactggat atgttgtgtt
     7561 ttacagtatt atgtagtctg ttttttatgc aaaatctaat ttaatatatt gatatttata
     7621 tcattttacg tttctcgttc agctttcttg tacaaagttg gcattataag aaagcattgc
     7681 ttatcaattt gttgcaacga acaggtcact atcagtcaaa ataaaatcat tatttgccat
     7741 ccagctga
//
