LOCUS       sequence-220447-        6855 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multisite (attR3/attR4) Gateway vector for expression in oomycetes. 
            Confers resistance to geneticin (G418)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6855)
  AUTHORS   Evangelisti E, Shenhav L, Yunusov T, Le Naour-Vernet M, Rink P, 
            Schornack S
  TITLE     Hydrodynamic Shape Changes Underpin Nuclear Rerouting in Branched 
            Hyphae of an Oomycete Pathogen.
  JOURNAL   MBio. 2019 Oct 1;10(5). pii: mBio.01516-19. doi: 
            10.1128/mBio.01516-19.
  PUBMED    31575765
REFERENCE   2  (bases 1 to 6855)
  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: "MBio."; 
            date: "2019-10-1"; volume: "10(5). pii: mBio.01516-19. doi"; pages: 
            " 10.1128/mBio.01516-19"
COMMENT     Sequence Label: pTORKm43GW
FEATURES             Location/Qualifiers
     source          1..6855
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(322..341)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(323..341)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(348..365)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(348..364)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(357..379)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(505..960)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(642..663)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     854..873
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        987..1091
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1092..1160,1161..1952)
                     /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(1310..1329)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2123..2711
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2612..2631
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2865..2882
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2999..3020
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3035..3040,3041..3058,3059..3065)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3073..3089
                     /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     3078..3100
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3097..3113
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3097..3113
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3134..3152
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    3340..3463
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(3504..3809)
                     /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="MQFKVFTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(3606..3625)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(4129..4809)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     4724..4743
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(4863..4869,4870..4887,4888..4893))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(4918..5041)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             5655..6449
                     /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(5709..5728)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6319..6338
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 tacgccgcct tagtaacaaa catgccaaaa gaaggaaagc tttcaccagc tttcaagttt
       61 aatcaatctt tgagagtcac aattgcaaga ataaatgaat ttttacaaca cagttcgatg
      121 cactgtgttt cgtcttgatg aaaattggac ggcaaatcga gtatcttaaa caaatctcgg
      181 cataggctgt ccattttgtg gtcgcctctt tgttggtagt ttgcagtagt ttcattagta
      241 gaagatctgc atcctcaatc tgctaaacgt cttgagcagc tatgagacga tccggggaat
      301 tggggcccga gctccaattc gccctatagt gagtcgtatt acaattcact ggccgtcgtt
      361 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
      421 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
      481 ttgcgcagcc tgaatggcga atggaaattg taagcgttaa tattttgtta aaattcgcgt
      541 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
      601 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
      661 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
      721 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
      781 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
      841 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
      901 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
      961 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     1021 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     1081 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     1141 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     1201 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     1261 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     1321 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     1381 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     1441 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     1501 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     1561 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     1621 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     1681 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     1741 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     1801 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     1861 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     1921 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     1981 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     2041 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     2101 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     2161 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     2221 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     2281 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     2341 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     2401 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     2461 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     2521 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     2581 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     2641 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     2701 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     2761 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     2821 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     2881 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     2941 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     3001 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
     3061 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
     3121 acgccaagct cgaaattaac cctcactaaa gggaacaaaa gctggtaccg ggcccattat
     3181 acctcgtatt cgattaaatg atacaactca ttgccacacc gcaagacggc gtcaccacca
     3241 tagacagcct ttcaagcacc cccttgtcta tgagctgcca cgtcagctgc cgctcctaat
     3301 atccacccgc gtccctattc ccgtcatatg ccaagctatc aactttgtat agaaaagttg
     3361 aacgagaaac gtaaaatgat ataaatatca atatattaaa ttagattttg cataaaaaac
     3421 agactacata atactgtaaa acacaacata tccagtcact atggtcgacc tgcagactgg
     3481 ctgtgtataa gggagcctga catttatatt ccccagaaca tcaggttaat ggcgtttttg
     3541 atgtcatttt cgcggtgact gagatcagcc acttcttccc cgataacgga gaccggcaca
     3601 ctggccatat cggtggtcat catgcgccag ctttcatccc cgatatgcac caccgggtaa
     3661 agttcacggg ggactttatc tgacagcaga cgtgcactgg ccagggggat caccatccgt
     3721 cgcccgggcg tgtcaataat atcactctgt acatccacaa acagacgata acggctctct
     3781 cttttatagg tgaaaacctt aaactgcatt tcaccagccc ctgttctcgt cggcaaaaga
     3841 gccgttcatt tcaataaacc gggcgacctc agccatccct tcttgatttt ccgctttcca
     3901 gcgttcggca cgcagacgac gggcttcatt ctgcatggtt gtgcttaccg aaccggagat
     3961 actgacatca tatatgcctt gagcaactga tagctgtcgc tgtcaactgt cactgtgata
     4021 cgctgcttca tagcatacct ctttttgaca tacttcgggt atacatatca gtatatattc
     4081 ttataccgca aaaatcagcg cgcaaatacg catactgtta tctggctttt agtaagccgg
     4141 atcctctaga ttacgccccg cctgccactc atcgcagtac tgttgtaatt cattaagcat
     4201 tctgccgaca tggaagccat cacaaacggc atgatgaacc tgaatcgcca gcggcatcag
     4261 caccttgtcg ccttgcgtat aatatttgcc catggtgaaa acgggggcga agaagttgtc
     4321 catattggcc acgtttaaat caaaactggt gaaactcacc cagggattgg ctgagacgaa
     4381 aaacatattc tcaataaacc ctttagggaa ataggccagg ttttcaccgt aacacgccac
     4441 atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg tggtattcac tccagagcga
     4501 tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa gggtgaacac tatcccatat
     4561 caccagctca ccgtctttca ttgccatacg gaattccgga tgagcattca tcaggcgggc
     4621 aagaatgtga ataaaggccg gataaaactt gtgcttattt ttctttacgg tctttaaaaa
     4681 ggccgtaata tccagctgaa cggtctggtt ataggtacat tgagcaactg actgaaatgc
     4741 ctcaaaatgt tctttacgat gccattggga tatatcaacg gtggtatatc cagtgatttt
     4801 tttctccatt ttagcttcct tagctcctga aaatctcgac ggatcctaac tcaaaatcca
     4861 cacattatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg cggccgccat
     4921 agtgactgga tatgttgtgt tttacagtat tatgtagtct gttttttatg caaaatctaa
     4981 tttaatatat tgatatttat atcattttac gtttctcgtt caactttatt atacatagtt
     5041 gataattcac atatgctcaa tgcaattgac tgtgtcctcg attatttcta ttatttaata
     5101 ttatttccat taaaaaagaa atccagttcg tggcaaaata agacaaaggt ttaaataaaa
     5161 atttatcgtt attaaactcg taacttacct tcttcatggc tcaacccaat ctgtatatcg
     5221 acttatttaa tttggttaac aaatcggttt tcgtcgcaaa taggggatga caactaaaat
     5281 accaatttgt gtccttgcga cacgtcaaca ggtggctgcc aatatctcag gttttgagca
     5341 cacaacgctg tgattggcta tctgtagatc gcttcgagaa ctttcggacc gacatatcag
     5401 aacgcagtca tgtccaatcc agctaacgat tggtcgaaac cgcacttcat cgcttcattt
     5461 caaccaattc ccatctacta aaacgagcgt cggaatagtc tcgaacgtaa ccccacgtag
     5521 tgggtggcag cgcccctcgt atctttcaat cgccactcga gtctcaagtt tgctcacttt
     5581 gaaattttcc atctggacta cacatcaaat ctaactcaac ttttatcaat tgaaacacaa
     5641 gggccccgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     5701 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     5761 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     5821 ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     5881 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     5941 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     6001 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     6061 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     6121 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     6181 cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     6241 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     6301 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     6361 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     6421 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
     6481 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
     6541 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     6601 gctggagttc ttcgcccacc ccccggatcc ccggggtcga ctagaatact tagcaattgg
     6661 ttagtttgaa aattgtcaaa gcagagtttt ttcgacaata aaatttattt tctaaatatt
     6721 tgatttagcg ttgacttaat ttgttgattc ggttgtcatc ttttgtgaag atgcccgaga
     6781 gtctagtaaa tctcaagcga aacatagtga gaaggtcact atggctggtt tgcatgagtc
     6841 tgggcctatt gtgct
//
