LOCUS       sequence-119643-        7480 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The multiple cloning site of pGreenII0000 cloned into binary vector 
            pDHt.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7480)
  AUTHORS   Khang CH, Park SY, Lee YH, Kang S
  TITLE     A dual selection based, targeted gene replacement tool for 
            Magnaporthe grisea and Fusarium oxysporum.
  JOURNAL   Fungal Genet Biol. 2005 Jun;42(6):483-92. Epub 2005 Apr 25.
  PUBMED    15893252
REFERENCE   2  (bases 1 to 7480)
  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: "Fungal 
            Genet Biol."; date: "2005-06"; volume: "42(6)"; pages: "483-92. Epub
            2005 Apr 25"
COMMENT     Sequence Label: pDHt-KS
FEATURES             Location/Qualifiers
     source          1..7480
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..25
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     polyA_signal    103..277
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     CDS             complement(360..725)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWSSTAVAAALELVDPPGCRNSISSLSI
                     PSTSRGGPVPNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
                     TDRPSQQLRSLNGEWKL"
     primer_bind     494..516
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     508..525
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     509..525
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     535..554
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        535..553
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    562..669
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     578..594
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     579..595
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(629..645)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(629..645)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(682..702)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(682..700)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(721..737)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(721..737)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(734..756)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    745..761
                     /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(769..775,776..793,794..799))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    814..835
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     misc_feature    1203..1227
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             2527..3156
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             3590..4657
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="GRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESWQA
                     AADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLSKR
                     DRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKPGR
                     VFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGEAL
                     ISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYRLA
                     RRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPILV
                     MRYRNLIEGEASAGS"
     rep_origin      4723..4917
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(5153..5175)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5261..5401
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5316..5335
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(5587..6175)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5667..5686)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6262..7056)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
ORIGIN
        1 tggcaggata tattgtggtg taaacaaatt gacgcttaga caacttaata acacattgcg
       61 gacgttttta atgtactgaa ttaacgccga attaattcgg gggatctgga ttttagtact
      121 ggattttggt tttaggaatt agaaatttta ttgatagaag tattttacaa atacaaatac
      181 atactaaggg tttcttatat gctcaacaca tgagcgaaac cctataggaa ccctaattcc
      241 cttatctggg aactactcac acattattat ggagaaactc gattggcaag ctgctctagc
      301 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcaaa cattaacgct
      361 tacaatttcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
      421 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
      481 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac
      541 gactcactat agggcgaatt gggtaccggg ccccccctcg aggtcgacgg tatcgataag
      601 cttgatatcg aattcctgca gcccggggga tccactagtt ctagagcggc cgccaccgcg
      661 gtggagctcc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg
      721 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
      781 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
      841 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
      901 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
      961 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     1021 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgaagg ctggcgtaat
     1081 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgc
     1141 tagagcagct tgagcttgga tcagattgtc gtttcccgcc ttcagtttaa actatcagtg
     1201 tttgacagga tatattggcg ggtaaaccta agagaaaaga gcgtttatta gaataacgga
     1261 tatttaaaag ggcgtgaaaa ggtttatccg ttcgtccatt tgtatgtgca tgccaaccac
     1321 agggttcccc tcgggatcaa agtactttga tccaacccct ccgctgctat agtgcagtcg
     1381 gcttctgacg ttcagtgcag ccgtcttctg aaaacgacat gtcgcacaag tcctaagtta
     1441 cgcgacaggc tgccgccctg cccttttcct ggcgttttct tgtcgcgtgt tttagtcgca
     1501 taaagtagaa tacttgcgac tagaaccgga gacattacgc catgaacaag agcgccgccg
     1561 ctggcctgct gggctatgcc cgcgtcagca ccgacgacca ggacttgacc aaccaacggg
     1621 ccgaactgca cgcggccggc tgcaccaagc tgttttccga gaagatcacc ggcaccaggc
     1681 gcgaccgccc ggagctggcc aggatgcttg accacctacg ccctggcgac gttgtgacag
     1741 tgaccaggct agaccgcctg gcccgcagca cccgcgacct actggacatt gccgagcgca
     1801 tccaggaggc cggcgcgggc ctgcgtagcc tggcagagcc gtgggccgac accaccacgc
     1861 cggccggccg catggtgttg accgtgttcg ccggcattgc cgagttcgag cgttccctaa
     1921 tcatcgaccg cacccggagc gggcgcgagg ccgccaaggc ccgaggcgtg aagtttggcc
     1981 cccgccctac cctcaccccg gcacagatcg cgcacgcccg cgagctgatc gaccaggaag
     2041 gccgcaccgt gaaagaggcg gctgcactgc ttggcgtgca tcgctcgacc ctgtaccgcg
     2101 cacttgagcg cagcgaggaa gtgacgccca ccgaggccag gcggcgcggt gccttccgtg
     2161 aggacgcatt gaccgaggcc gacgccctgg cggccgccga gaatgaacgc caagaggaac
     2221 aagcatgaaa ccgcaccagg acggccagga cgaaccgttt ttcattaccg aagagatcga
     2281 ggcggagatg atcgcggccg ggtacgtgtt cgagccgccc gcgcacgtct caaccgtgcg
     2341 gctgcatgaa atcctggccg gtttgtctga tgccaagctg gcggcctggc cggccagctt
     2401 ggccgctgaa gaaaccgagc gccgccgtct aaaaaggtga tgtgtatttg agtaaaacag
     2461 cttgcgtcat gcggtcgctg cgtatatgat gcgatgagta aataaacaaa tacgcaaggg
     2521 gaacgcatga aggttatcgc tgtacttaac cagaaaggcg ggtcaggcaa gacgaccatc
     2581 gcaacccatc tagcccgcgc cctgcaactc gccggggccg atgttctgtt agtcgattcc
     2641 gatccccagg gcagtgcccg cgattgggcg gccgtgcggg aagatcaacc gctaaccgtt
     2701 gtcggcatcg accgcccgac gattgaccgc gacgtgaagg ccatcggccg gcgcgacttc
     2761 gtagtgatcg acggagcgcc ccaggcggcg gacttggctg tgtccgcgat caaggcagcc
     2821 gacttcgtgc tgattccggt gcagccaagc ccttacgaca tatgggccac cgccgacctg
     2881 gtggagctgg ttaagcagcg cattgaggtc acggatggaa ggctacaagc ggcctttgtc
     2941 gtgtcgcggg cgatcaaagg cacgcgcatc ggcggtgagg ttgccgaggc gctggccggg
     3001 tacgagctgc ccattcttga gtcccgtatc acgcagcgcg tgagctaccc aggcactgcc
     3061 gccgccggca caaccgttct tgaatcagaa cccgagggcg acgctgcccg cgaggtccag
     3121 gcgctggccg ctgaaattaa atcaaaactc atttgagtta atgaggtaaa gagaaaatga
     3181 gcaaaagcac aaacacgcta agtgccggcc gtccgagcgc acgcagcagc aaggctgcaa
     3241 cgttggccag cctggcagac acgccagcca tgaagcgggt caactttcag ttgccggcgg
     3301 aggatcacac caagctgaag atgtacgcgg tacgccaagg caagaccatt accgagctgc
     3361 tatctgaata catcgcgcag ctaccagagt aaatgagcaa atgaataaat gagtagatga
     3421 attttagcgg ctaaaggagg cggcatggaa aatcaagaac aaccaggcac cgacgccgtg
     3481 gaatgcccca tgtgtggagg aacgggcggt tggccaggcg taagcggctg ggttgtctgc
     3541 cggccctgca atggcactgg aacccccaag cccgaggaat cggcgtgacg gtcgcaaacc
     3601 atccggcccg gtacaaatcg gcgcggcgct gggtgatgac ctggtggaga agttgaaggc
     3661 cgcgcaggcc gcccagcggc aacgcatcga ggcagaagca cgccccggtg aatcgtggca
     3721 agcggccgct gatcgaatcc gcaaagaatc ccggcaaccg ccggcagccg gtgcgccgtc
     3781 gattaggaag ccgcccaagg gcgacgagca accagatttt ttcgttccga tgctctatga
     3841 cgtgggcacc cgcgatagtc gcagcatcat ggacgtggcc gttttccgtc tgtcgaagcg
     3901 tgaccgacga gctggcgagg tgatccgcta cgagcttcca gacgggcacg tagaggtttc
     3961 cgcagggccg gccggcatgg ccagtgtgtg ggattacgac ctggtactga tggcggtttc
     4021 ccatctaacc gaatccatga accgataccg ggaagggaag ggagacaagc ccggccgcgt
     4081 gttccgtcca cacgttgcgg acgtactcaa gttctgccgg cgagccgatg gcggaaagca
     4141 gaaagacgac ctggtagaaa cctgcattcg gttaaacacc acgcacgttg ccatgcagcg
     4201 tacgaagaag gccaagaacg gccgcctggt gacggtatcc gagggtgaag ccttgattag
     4261 ccgctacaag atcgtaaaga gcgaaaccgg gcggccggag tacatcgaga tcgagctagc
     4321 tgattggatg taccgcgaga tcacagaagg caagaacccg gacgtgctga cggttcaccc
     4381 cgattacttt ttgatcgatc ccggcatcgg ccgttttctc taccgcctgg cacgccgcgc
     4441 cgcaggcaag gcagaagcca gatggttgtt caagacgatc tacgaacgca gtggcagcgc
     4501 cggagagttc aagaagttct gtttcaccgt gcgcaagctg atcgggtcaa atgacctgcc
     4561 ggagtacgat ttgaaggagg aggcggggca ggctggcccg atcctagtca tgcgctaccg
     4621 caacctgatc gagggcgaag catccgccgg ttcctaatgt acggagcaga tgctagggca
     4681 aattgcccta gcaggggaaa aaggtcgaaa aggtctcttt cctgtggata gcacgtacat
     4741 tgggaaccca aagccgtaca ttgggaaccg gaacccgtac attgggaacc caaagccgta
     4801 cattgggaac cggtcacaca tgtaagtgac tgatataaaa gagaaaaaag gcgatttttc
     4861 cgcctaaaac tctttaaaac ttattaaaac tcttaaaacc cgcctggcct gtgcataact
     4921 gtctggccag cgcacagccg aagagctgca aaaagcgcct acccttcggt cgctgcgctc
     4981 cctacgcccc gccgcttcgc gtcggcctat cgcggccgct ggccgctcaa aaatggctgg
     5041 cctacggcca ggcaatctac cagggcgcgg acaagccgcg ccgtcgccac tcgaccgccg
     5101 gcgcccacat caaggcaccc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac
     5161 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
     5221 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac
     5281 gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag
     5341 agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
     5401 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     5461 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     5521 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     5581 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     5641 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     5701 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     5761 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     5821 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     5881 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     5941 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     6001 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
     6061 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     6121 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     6181 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     6241 tttggtcatg cattctaggt actaaaacaa ttcatccagt aaaatataat attttatttt
     6301 ctcccaatca ggcttgatcc ccagtaagtc aaaaaatagc tcgacatact gttcttcccc
     6361 gatatcctcc ctgatcgacc ggacgcagaa ggcaatgtca taccacttgt ccgccctgcc
     6421 gcttctccca agatcaataa agccacttac tttgccatct ttcacaaaga tgttgctgtc
     6481 tcccaggtcg ccgtgggaaa agacaagttc ctcttcgggc ttttccgtct ttaaaaaatc
     6541 atacagctcg cgcggatctt taaatggagt gtcttcttcc cagttttcgc aatccacatc
     6601 ggccagatcg ttattcagta agtaatccaa ttcggctaag cggctgtcta agctattcgt
     6661 atagggacaa tccgatatgt cgatggagtg aaagagcctg atgcactccg catacagctc
     6721 gataatcttt tcagggcttt gttcatcttc atactcttcc gagcaaagga cgccatcggc
     6781 ctcactcatg agcagattgc tccagccatc atgccgttca aagtgcagga cctttggaac
     6841 aggcagcttt ccttccagcc atagcatcat gtccttttcc cgttccacat cataggtggt
     6901 ccctttatac cggctgtccg tcatttttaa atataggttt tcattttctc ccaccagctt
     6961 atatacctta gcaggagaca ttccttccgt atcttttacg cagcggtatt tttcgatcag
     7021 ttttttcaat tccggtgata ttctcatttt agccatttat tatttccttc ctcttttcta
     7081 cagtatttaa agatacccca agaagctaat tataacaaga cgaactccaa ttcactgttc
     7141 cttgcattct aaaaccttaa ataccagaaa acagcttttt caaagttgtt ttcaaagttg
     7201 gcgtataaca tagtatcgac ggagccgatt ttgaaaccgc ggtgatcaca ggcagcaacg
     7261 ctctgtcatc gttacaatca acatgctacc ctccgcgaga tcatccgtgt ttcaaacccg
     7321 gcagcttagt tgccgttctt ccgaatagca tcggtaacat gagcaaagtc tgccgcctta
     7381 caacggctct cccgctgacg ccgtcccgga ctgatgggct gcctgtatcg agtggtgatt
     7441 ttgtgccgag ctgccggtcg gggagctgtt ggctggctgg
//
