LOCUS       sequence-220568-        8429 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multisite (attR3/attR4) Gateway vector for expression in oomycetes. 
            Confers resistance to gentamicin. Contains additional cassette for 
            constitutive expression of mWasabi..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8429)
  AUTHORS   Evangelisti E, Yunusov T, Shenhav L, Schornack S
  TITLE     N-acetyltransferase AAC(3)-I confers gentamicin resistance to 
            Phytophthora palmivora and Phytophthora infestans.
  JOURNAL   BMC Microbiol. 2019 Nov 27;19(1):265. doi: 
            10.1186/s12866-019-1642-0.
  PUBMED    31775609
REFERENCE   2  (bases 1 to 8429)
  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: "BMC 
            Microbiol."; date: "2019-11-27"; volume: "19(1):265. doi"; pages: " 
            10.1186/s12866-019-1642-0"
COMMENT     Sequence Label: pTORGm43GWF
FEATURES             Location/Qualifiers
     source          1..8429
                     /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"
     primer_bind     3745..3761
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     3746..3762
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             3768..4478
                     /codon_start=1
                     /product="green variant of teal fluorescent protein mTFP1 
                     (Ai et al., 2008)"
                     /label=mWasabi
                     /translation="MVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDG
                     TNTINLEVKEGAPLPFSYDILTTAFSYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTF
                     EDKGIVKVKSDISMEEYSFIYEIHLKGENFPPNGPVMQKETTGWDASTERMYVRDGVLK
                     GDVKMKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYEIA
                     VARNSTDGMDELYK"
     protein_bind    5175..5298
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(5339..5644)
                     /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(5441..5460)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(5964..6644)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     6559..6578
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(6698..6704,6705..6722,6723..6728))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(6753..6876)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             7490..8023
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
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 gctggtacct ctgatggaca
     3181 aagggtcgcc tctacgtacc cgcgacaatc tcggcttttc caatgtcgac agctggcata
     3241 tcgcacgcga ggttaggcgt cttcagccgg cgtaaccaca aacctcgagt caagtaccct
     3301 ttttcagacc ttaatttccg tgtccattcc ctcgcgttta cacgaaggtt tctgagaaat
     3361 accacatgtc acagagagag agaagattgg agaaccttct caacctgacc cgtgcagagc
     3421 gcttttgcgt cctaccatcc gttaattata gcaccaccac cacatgtctc gccggaactc
     3481 tcttatccgc tctttttgat cgtctttttt gcgccccatt aaagacatct cttgcacagg
     3541 gatcgtttct gattggccat ttcacttcct cttttacatc cgaaaaccac ctgtttgtgg
     3601 ggttgtgaag ccacaaccgg acgctcaaaa agcctcgccc gactcgccca cgatcggaag
     3661 gctcattctc cttttcactc tcacgtgccc aagtcccaac cgactctttt tcgaccttca
     3721 ctctcaccga caacgggccc cccctcgagg tcgacggtat cgataccatg gtgagcaagg
     3781 gcgaggagac cacaatgggc gtaatcaagc ccgacatgaa gatcaagctg aagatggagg
     3841 gcaacgtgaa tggccacgcc ttcgtgatcg agggcgaggg cgagggcaag ccctacgacg
     3901 gcaccaacac catcaacctg gaggtgaagg agggagcccc cctgcccttc tcctacgaca
     3961 ttctgaccac cgcgttcagt tacggcaaca gggccttcac caagtacccc gacgacatcc
     4021 ccaactactt caagcagtcc ttccccgagg gctactcttg ggagcgcacc atgaccttcg
     4081 aggacaaggg catcgtgaag gtgaagtccg acatctccat ggaggagtac tccttcatct
     4141 acgagataca cctcaagggc gagaacttcc cccccaacgg ccccgtgatg cagaaggaga
     4201 ccaccggctg ggacgcctcc accgagagga tgtacgtgcg cgacggcgtg ctgaagggcg
     4261 acgtcaagat gaagctgctg ctggagggcg gcggccacca ccgcgttgac ttcaagacca
     4321 tctacagggc caagaaggcg gtgaagctgc ccgactatca ctttgtggac caccgcatcg
     4381 agatcctgaa ccacgacaag gactacaaca aggtgaccgt ttacgagatc gccgtggccc
     4441 gcaactccac cgacggcatg gacgagctgt acaagtaggc cgaaggtcga gctgtgtgca
     4501 ctgagtgcga agttgcagat tttgtgtcta tgcatttaat acaatttcct tttttccatt
     4561 ttcgttgttt tttttgtgaa agaacgattg ttttcaaaag aaggccgttt gtagaacaat
     4621 ttttttttat actagcattt ggtattcgag tgtgacgaat cggtgatggc gctttacaaa
     4681 tgaaatataa cgctgaacaa gcgcagtgac agctttattt tataacagag aataaagacc
     4741 aaagtagagt agactgatgg cggcttgaaa caaaactacg tgacttctaa tgcaaagaaa
     4801 accatacccc acgctactta gaattgtttc tctttggttg atgaccgcta tagtcgtatg
     4861 gagtgatccc tttttgtcaa tatttcgatt cttgctaaaa ccgtgactat tttgaggcaa
     4921 ctgtgtgtac ttaacatggt agaaatttct atgtttaagc cagctgacaa tcattcttta
     4981 tttatcgtaa accaaccagg taccgggccc attatacctc gtattcgatt aaatgataca
     5041 actcattgcc acaccgcaag acggcgtcac caccatagac agcctttcaa gcaccccctt
     5101 gtctatgagc tgccacgtca gctgccgctc ctaatatcca cccgcgtccc tattcccgtc
     5161 atatgccaag ctatcaactt tgtatagaaa agttgaacga gaaacgtaaa atgatataaa
     5221 tatcaatata ttaaattaga ttttgcataa aaaacagact acataatact gtaaaacaca
     5281 acatatccag tcactatggt cgacctgcag actggctgtg tataagggag cctgacattt
     5341 atattcccca gaacatcagg ttaatggcgt ttttgatgtc attttcgcgg tgactgagat
     5401 cagccacttc ttccccgata acggagaccg gcacactggc catatcggtg gtcatcatgc
     5461 gccagctttc atccccgata tgcaccaccg ggtaaagttc acgggggact ttatctgaca
     5521 gcagacgtgc actggccagg gggatcacca tccgtcgccc gggcgtgtca ataatatcac
     5581 tctgtacatc cacaaacaga cgataacggc tctctctttt ataggtgaaa accttaaact
     5641 gcatttcacc agcccctgtt ctcgtcggca aaagagccgt tcatttcaat aaaccgggcg
     5701 acctcagcca tcccttcttg attttccgct ttccagcgtt cggcacgcag acgacgggct
     5761 tcattctgca tggttgtgct taccgaaccg gagatactga catcatatat gccttgagca
     5821 actgatagct gtcgctgtca actgtcactg tgatacgctg cttcatagca tacctctttt
     5881 tgacatactt cgggtataca tatcagtata tattcttata ccgcaaaaat cagcgcgcaa
     5941 atacgcatac tgttatctgg cttttagtaa gccggatcct ctagattacg ccccgcctgc
     6001 cactcatcgc agtactgttg taattcatta agcattctgc cgacatggaa gccatcacaa
     6061 acggcatgat gaacctgaat cgccagcggc atcagcacct tgtcgccttg cgtataatat
     6121 ttgcccatgg tgaaaacggg ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa
     6181 ctggtgaaac tcacccaggg attggctgag acgaaaaaca tattctcaat aaacccttta
     6241 gggaaatagg ccaggttttc accgtaacac gccacatctt gcgaatatat gtgtagaaac
     6301 tgccggaaat cgtcgtggta ttcactccag agcgatgaaa acgtttcagt ttgctcatgg
     6361 aaaacggtgt aacaagggtg aacactatcc catatcacca gctcaccgtc tttcattgcc
     6421 atacggaatt ccggatgagc attcatcagg cgggcaagaa tgtgaataaa ggccggataa
     6481 aacttgtgct tatttttctt tacggtcttt aaaaaggccg taatatccag ctgaacggtc
     6541 tggttatagg tacattgagc aactgactga aatgcctcaa aatgttcttt acgatgccat
     6601 tgggatatat caacggtggt atatccagtg atttttttct ccattttagc ttccttagct
     6661 cctgaaaatc tcgacggatc ctaactcaaa atccacacat tatacgagcc ggaagcataa
     6721 agtgtaaagc ctggggtgcc taatgcggcc gccatagtga ctggatatgt tgtgttttac
     6781 agtattatgt agtctgtttt ttatgcaaaa tctaatttaa tatattgata tttatatcat
     6841 tttacgtttc tcgttcaact ttattataca tagttgataa ttcacatatg ctcaatgcaa
     6901 ttgactgtgt cctcgattat ttctattatt taatattatt tccattaaaa aagaaatcca
     6961 gttcgtggca aaataagaca aaggtttaaa taaaaattta tcgttattaa actcgtaact
     7021 taccttcttc atggctcaac ccaatctgta tatcgactta tttaatttgg ttaacaaatc
     7081 ggttttcgtc gcaaataggg gatgacaact aaaataccaa tttgtgtcct tgcgacacgt
     7141 caacaggtgg ctgccaatat ctcaggtttt gagcacacaa cgctgtgatt ggctatctgt
     7201 agatcgcttc gagaactttc ggaccgacat atcagaacgc agtcatgtcc aatccagcta
     7261 acgattggtc gaaaccgcac ttcatcgctt catttcaacc aattcccatc tactaaaacg
     7321 agcgtcggaa tagtctcgaa cgtaacccca cgtagtgggt ggcagcgccc ctcgtatctt
     7381 tcaatcgcca ctcgagtctc aagtttgctc actttgaaat tttccatctg gactacacat
     7441 caaatctaac tcaactttta tcaattgaaa cacaagggcc ccgtttcgca tgttacgcag
     7501 cagcaacgat gttacgcagc agggcagtcg ccctaaaaca aagttaggtg gctcaagtat
     7561 gggcatcatt cgcacatgta ggctcggccc tgaccaagtc aaatccatgc gggctgctct
     7621 tgatcttttc ggtcgtgagt tcggagacgt agccacctac tcccaacatc agccggactc
     7681 cgattacctc gggaacttgc tccgtagtaa gacattcatc gcgcttgctg ccttcgacca
     7741 agaagcggtt gttggcgctc tcgcggctta cgttctgccc aagtttgagc agccgcgtag
     7801 tgagatctat atctatgatc tcgcagtctc cggcgagcac cggaggcagg gcattgccac
     7861 cgcgctcatc aatctcctca agcatgaggc caacgcgctt ggtgcttatg tgatctacgt
     7921 gcaagcagat tacggtgacg atcccgcagt ggctctctat acaaagttgg gcatacggga
     7981 agaagtgatg cactttgata tcgacccaag taccgccacc taagcgggac tctggggttc
     8041 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     8101 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     8161 cgcggggatc tcatgctgga gttcttcgcc caccccccgg atccccgggg tcgactagaa
     8221 tacttagcaa ttggttagtt tgaaaattgt caaagcagag ttttttcgac aataaaattt
     8281 attttctaaa tatttgattt agcgttgact taatttgttg attcggttgt catcttttgt
     8341 gaagatgccc gagagtctag taaatctcaa gcgaaacata gtgagaaggt cactatggct
     8401 ggtttgcatg agtctgggcc tattgtgct
//
