LOCUS       sequence-390013-        7827 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A yeast-Escherichia coli-Agrobacterium shuttle vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7827)
  AUTHORS   Sun K, Zhao D, Liu Y, Huang C, Zhang W, Li Z
  TITLE     Rapid Construction of Complex Plant RNA Virus Infectious cDNA Clones
            for Agroinfection Using a Yeast-E. coli-Agrobacterium Shuttle 
            Vector.
  JOURNAL   Viruses. 2017 Nov 7;9(11):332. doi: 10.3390/v9110332.
  PUBMED    29112135
REFERENCE   2  (bases 1 to 7827)
  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: "Viruses."; 
            date: "2017-11-7"; volume: "9(11):332. doi"; pages: " 
            10.3390/v9110332"
COMMENT     Sequence Label: pCB301-2u-HDV
FEATURES             Location/Qualifiers
     source          1..7827
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     711..731
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     promoter        720..765
                     /label=minimal CaMV 35S promoter
                     /note="minimal 35S promoter from cauliflower mosaic virus"
     terminator      896..1148
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     misc_feature    1196..1220
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      1348..2690
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(2311..2358)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     primer_bind     2731..2749
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2787..2809)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2909..2928
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2949..3230
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             3231..3905
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     rep_origin      complement(4008..4463)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4145..4166)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4357..4376
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     CDS             complement(4579..5727)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MAQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     CDS             complement(6026..6820)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHLERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      7029..7660
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    7740..7764
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 aagcttgcat gcctgcaggt caacatggtg gagcacgaca cacttgtcta ctccaaaaat
       61 atcaaagata cagtctcaga agaccaaagg gcaattgaga cttttcaaca aagggtaata
      121 tccggaaacc tcctcggatt ccattgccca gctatctgtc actttattgt gaagatagtg
      181 gaaaaggaag gtggctccta caaatgccat cattgcgata aaggaaaggc catcgttgaa
      241 gatgcctctg ccgacagtgg tcccaaagat ggacccccac ccacgaggag catcgtggaa
      301 aaagaagacg ttccaaccac gtcttcaaag caagtggatt gatgtgataa catggtggag
      361 cacgacacac ttgtctactc caaaaatatc aaagatacag tctcagaaga ccaaagggca
      421 attgagactt ttcaacaaag ggtaatatcc ggaaacctcc tcggattcca ttgcccagct
      481 atctgtcact ttattgtgaa gatagtggaa aaggaaggtg gctcctacaa atgccatcat
      541 tgcgataaag gaaaggccat cgttgaagat gcctctgccg acagtggtcc caaagatgga
      601 cccccaccca cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa
      661 gtggattgat gtgatatctc cactgacgta agggatgacg cacaatccca ctatccttcg
      721 caagaccctt cctctatata aggaagttca tttcatttgg agaggcctga cctgcaggtc
      781 gactctagag gatccccggg tcggcatggc atctccacct cctcgcggtc cgacctgggc
      841 atccgaagga ggacgtcgtc cactcggatg gctaagggag agctcgaatt tccccgatcg
      901 ttcaaacatt tggcaataaa gtttcttaag attgaatcct gttgccggtc ttgcgatgat
      961 tatcatataa tttctgttga attacgttaa gcatgtaata attaacatgt aatgcatgac
     1021 gttatttatg agatgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat
     1081 agaaaacaaa atatagcgcg caaactagga taaattatcg cgcgcggtgt catctatgtt
     1141 actagatcgg aattcagatt gtcgtttccc gccttcagtt taaactatca gtgtttgaca
     1201 ggatatattg gcgggtaaac ctaagagaaa agagcgttta ttagaataat cggatattta
     1261 aaagggcgtg aaaaggttta tccgttcgtc catttgtatg tgcatgccaa ccacaggaga
     1321 tctcagtaaa gcgaaaagtg ccacctgaac gaagcatctg tgcttcattt tgtagaacaa
     1381 aaatgcaacg cgagagcgct aatttttcaa acaaagaatc tgagctgcat ttttacagaa
     1441 cagaaatgca acgcgaaagc gctattttac caacgaagaa tctgtgcttc atttttgtaa
     1501 aacaaaaatg caacgcgaga gcgctaattt ttcaaacaaa gaatctgagc tgcattttta
     1561 cagaacagaa atgcaacgcg agagcgctat tttaccaaca aagaatctat acttcttttt
     1621 tgttctacaa aaatgcatcc cgagagcgct atttttctaa caaagcatct tagattactt
     1681 tttttctcct ttgtgcgctc tataatgcag tctcttgata actttttgca ctgtaggtcc
     1741 gttaaggtta gaagaaggct actttggtgt ctattttctc ttccataaaa aaagcctgac
     1801 tccacttccc gcgtttactg attactagcg aagctgcggg tgcatttttt caagataaag
     1861 gcatccccga ttatattcta taccgatgtg gattgcgcat actttgtgaa cagaaagtga
     1921 tagcgttgat gattcttcat tggtcagaaa attatgaacg gtttcttcta ttttgtctct
     1981 atatactacg tataggaaat gtttacattt tcgtattgtt ttcgattcac tctatgaata
     2041 gttcttacta caattttttt gtctaaagag taatactaga gataaacata aaaaatgtag
     2101 aggtcgagtt tagatgcaag ttcaaggagc gaaaggtgga tgggtaggtt atatagggat
     2161 atagcacaga gatatatagc aaagagatac ttttgagcaa tgtttgtgga agcggtattc
     2221 gcaatatttt agtagctcgt tacagtccgg tgcgtttttg gttttttgaa agtgcgtctt
     2281 cagagcgctt ttggttttca aaagcgctct gaagttccta tactttctag agaataggaa
     2341 cttcggaata ggaacttcaa agcgtttccg aaaacgagcg cttccgaaaa tgcaacgcga
     2401 gctgcgcaca tacagctcac tgttcacgtc gcacctatat ctgcgtgttg cctgtatata
     2461 tatatacatg agaagaacgg catagtgcgt gtttatgctt aaatgcgtac ttatatgcgt
     2521 ctatttatgt aggatgaaag gtagtctagt acctcctgtg atattatccc attccatgcg
     2581 gggtatcgta tgcttccttc agcactaccc tttagctgtt ctatatgctg ccactcctca
     2641 attggattag tctcatcctt caatgctatc atttcctttg atattggatc atactaagaa
     2701 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc
     2761 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca
     2821 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
     2881 ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac
     2941 catagatcaa cgacattact atatatataa tataggaagc atttaataga acagcatcgt
     3001 aatatatgtg tactttgcag ttatgacgcc agatggcagt agtggaagat attctttatt
     3061 gaaaaatagc ttgtcacctt acgtacaatc ttgatccgga gcttttcttt ttttgccgat
     3121 taagaattaa ttcggtcgaa aaaagaaaag gagagggcca agagggaggg cattggtgac
     3181 tattgagcac gtgagtatac gtgattaagc acacaaaggc agcttggagt atgtctgtta
     3241 ttaatttcac aggtagttct ggtccattgg tgaaagtttg cggcttgcag agcacagagg
     3301 ccgcagaatg tgctctagat tccgatgctg acttgctggg tattatatgt gtgcccaata
     3361 gaaagagaac aattgacccg gttattgcaa ggaaaatttc aagtcttgta aaagcatata
     3421 aaaatagttc aggcactccg aaatacttgg ttggcgtgtt tcgtaatcaa cctaaggagg
     3481 atgttttggc tctggtcaat gattacggca ttgatatcgt ccaactgcat ggagatgagt
     3541 cgtggcaaga ataccaagag ttcctcggtt tgccagttat taaaagactc gtatttccaa
     3601 aagactgcaa catactactc agtgcagctt cacagaaacc tcattcgttt attcccttgt
     3661 ttgattcaga agcaggtggg acaggtgaac ttttggattg gaactcgatt tctgactggg
     3721 ttggaaggca agagagcccc gaaagcttac attttatgtt agctggtgga ctgacgccag
     3781 aaaatgttgg tgatgcgctt agattaaatg gcgttattgg tgttgatgta agcggaggtg
     3841 tggagacaaa tggtgtaaaa gactctaaca aaatagcaaa tttcgtcaaa aatgctaaga
     3901 aataggttat tactgagtag tatttattta agtattgttt gtgcacttgc cgatctatgc
     3961 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggaaa ttgtaagcgt
     4021 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     4081 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     4141 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     4201 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     4261 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     4321 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     4381 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     4441 taatgcgccg ctacagggcg cgtccattcg ccattcaggc tgcgcaactg ttgggaaggg
     4501 cgatcggtgc gggcctcttc gctattacgc cagatccttg ctggctgaac ccccagccgg
     4561 aactgacccc acaaggccct agcgtttgca atgcaccagg tcatcattga cccaggcgtg
     4621 ttccaccagg ccgctgcctc gcaactcttc gcaggcttcg ccgacctgct cgcgccactt
     4681 cttcacgcgg gtggaatccg atccgcacat gaggcggaag gtttccagct tgagcgggta
     4741 cggctcccgg tgcgagctga aatagtcgaa catccgtcgg gccgtcggcg acagcttgcg
     4801 gtacttctcc catatgaatt tcgtgtagtg gtcgccagca aacagcacga cgatttcctc
     4861 gtcgatcagg acctggcaac gggacgtttt cttgccacgg tccaggacgc ggaagcggtg
     4921 cagcagcgac accgattcca ggtgcccaac gcggtcggac gtgaagccca tcgccgtcgc
     4981 ctgtaggcgc gacaggcatt cctcggcctt cgtgtaatac cggccattga tcgaccagcc
     5041 caggtcctgg caaagctcgt agaacgtgaa ggtgatcggc tcgccgatag gggtgcgctt
     5101 cgcgtactcc aacacctgct gccacaccag ttcgtcatcg tcggcccgca gctcgacgcc
     5161 ggtgtaggtg atcttcacgt ccttgttgac gtggaaaatg accttgtttt gcagcgcctc
     5221 gcgcgggatt ttcttgttgc gcgtggtgaa cagggcagag cgggccgtgt cgtttggcat
     5281 cgctcgcatc gtgtccggcc acggcgcaat atcgaacaag gaaagctgca tttccttgat
     5341 ctgctgcttc gtgtgtttca gcaacgcggc ctgcttggcc tcgctgacct gttttgccag
     5401 gtcctcgccg gcggtttttc gcttcttggt cgtcatagtt cctcgcgtgt cgatggtcat
     5461 cgacttcgcc aaacctgccg cctcctgttc gagacgacgc gaacgctcca cggcggccga
     5521 tggcgcgggc agggcagggg gagccagttg cacgctgtcg cgctcgatct tggccgtagc
     5581 ttgctgggcc atcgagccga cggactggaa ggtttcgcgg ggcgcacgca tgacggtgcg
     5641 gcttgcgatg gtttcggcat cctcggcgga aaaccccgcg tcgatcagtt cttgcctgta
     5701 tgccttccgg tcaaacgtcc gattcattca ccctccttgc gggattgccc cgactcacgc
     5761 cggggcaatg tgcccttatt cctgatttga cccgcctggt gccttggtgt ccagataatc
     5821 caccttatcg gcaatgaagt cggtcccgta gaccgtctgg ccgtccttct cgtacttggt
     5881 attccgaatc ttgccctgca cgaataccag cgaccccttg cccaaatact tgccgtgggc
     5941 ctcggcctga gagccaaaac acttgatgcg gaagaagtcg gtgcgctcct gcttgtcgcc
     6001 ggcatcgttg cgccacatct aggtactaaa acaattcatc cagtaaaata taatatttta
     6061 ttttctccca atcaggcttg atccccagta agtcaaaaaa tagctcgaca tactgttctt
     6121 ccccgatatc ctccctgatc gaccggacgc agaaggcaat gtcataccac ttgtccgccc
     6181 tgccgcttct cccaagatca ataaagccac ttactttgcc atctttcaca aagatgttgc
     6241 tgtctcccag gtcgccgtgg gaaaagacaa gttcctcttc gggcttttcc gtctttaaaa
     6301 aatcatacag ctcgcgcgga tctttaaatg gggtgtcttc ttcccagttt tcgcaatcca
     6361 catcggccag atcgttattc agtaagtaat ccaattcggc taagcggctg tctaagctat
     6421 tcgtataggg acaatccgat atgtcgatgg agtgaaagag cctgatgcac tccgcataca
     6481 gctcgataat cttttcaggg ctttgttcat cttcatactc ttccgagcaa aggacgccat
     6541 cggcctcact catgagcaga ttgctccagc catcatgccg ttcaaggtgc aggacctttg
     6601 gaacaggcag ctttccttcc agccatagca tcatgtcctt ttcccgttcc acatcatagg
     6661 tggtcccttt ataccggctg tccgtcattt ttaaatatag gttttcattt tctcccacca
     6721 gcttatatac cttagcagga gacattcctt ccgtatcttt tacgcagcgg tatttttcga
     6781 tcagtttttt caattccggt gatattctca ttttagccat ttattatttc cttcctcttt
     6841 tctacagtat ttaaagatac cccaagaagc taattataac aagacgaact ccaattcact
     6901 gttccttgca ttctaaaact ttaaatacca gaaaacagct ttttcaaagt tgttttcaaa
     6961 gttggcgtat aacatagtat cgacggagcc gattttgaaa ccacaattat gggtgatgct
     7021 gcaactcgag cgggccggga gggttcgaga agggggggca ccccccttcg gcgtgcgcgg
     7081 tcacgcgcac agggcgcagc cctggttaaa aacaaggttt ataaatattg gtttaaaagc
     7141 aggttaaaag acaggttagc ggtggccgaa aaacgggcgg aaacccttgc aaatgctgga
     7201 ttttctgcct gtggacagcc cctcaaatgt caataggtgc gcccctcatc tgtcagcact
     7261 ctgcccctca agtgtcaagg atcgcgcccc tcatctgtca gtagtcgcgc ccctcaagtg
     7321 tcaataccgc agggcactta tccccaggct tgtccacatc atctgtggga aactcgcgta
     7381 aaatcaggcg ttttcgccga tttgcgaggc tggccagctc cacgtcgccg gccgaaatcg
     7441 agcctgcccc tcatctgtca acgccgcgcc gggtgagtcg gcccctcaag tgtcaacgtc
     7501 cgcccctcat ctgtcagtga gggccaagtt ttccgcgagg tatccacaac gccggcggcc
     7561 gcggtgtctc gcacacggct tcgacggcgt ttctggcgcg tttgcagggc catagacggc
     7621 cgccagccca gcggcgaggg caaccagccc ggtgagcgtc tctagtggac tgatgggctg
     7681 cctgtatcga gtggtgattt tgtgccgagc tgccggtcgg ggagctgttg gctggctggt
     7741 ggcaggatat attgtggtgt aaacaaattg acgcttagac aacttaataa cacattgcgg
     7801 acgtttttaa tgtactgggg tggtttt
//
