LOCUS       sequence-119641-        7481 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 7481)
  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 7481)
  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-SK
FEATURES             Location/Qualifiers
     source          1..7481
                     /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"
     protein_bind    585..606
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(621..626,627..644,645..651)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    659..675
                     /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     664..686
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     683..699
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     683..699
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             695..1060
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWSSTAVAAALELVDPPGCRNSISSLSI
                     PSTSRGGPVPNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
                     TDRPSQQLRSLNGEWKL"
     primer_bind     718..738
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        720..738
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    751..858
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     775..791
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     775..791
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(825..841)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(826..842)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(866..885)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(867..885)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(895..912)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(895..911)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(904..926)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     misc_feature    1204..1228
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             2528..3157
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             3591..4658
                     /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      4724..4918
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(5154..5176)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5262..5402
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5317..5336
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(5588..6176)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5668..5687)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6263..7057)
                     /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 ttaatgcagc cttcatgttc
      361 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
      421 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
      481 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
      541 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
      601 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
      661 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca
      721 attaaccctc actaaaggga acaaaagctg gagctccacc gcggtggcgg ccgctctaga
      781 actagtggat cccccgggct gcaggaattc gatatcaagc ttatcgatac cgtcgacctc
      841 gagggggggc ccggtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc
      901 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
      961 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     1021 ccaacagttg cgcagcctga atggcgaatg gaaattgtaa gcgttaatgt tctggcgtaa
     1081 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     1141 ctagagcagc ttgagcttgg atcagattgt cgtttcccgc cttcagttta aactatcagt
     1201 gtttgacagg atatattggc gggtaaacct aagagaaaag agcgtttatt agaataacgg
     1261 atatttaaaa gggcgtgaaa aggtttatcc gttcgtccat ttgtatgtgc atgccaacca
     1321 cagggttccc ctcgggatca aagtactttg atccaacccc tccgctgcta tagtgcagtc
     1381 ggcttctgac gttcagtgca gccgtcttct gaaaacgaca tgtcgcacaa gtcctaagtt
     1441 acgcgacagg ctgccgccct gcccttttcc tggcgttttc ttgtcgcgtg ttttagtcgc
     1501 ataaagtaga atacttgcga ctagaaccgg agacattacg ccatgaacaa gagcgccgcc
     1561 gctggcctgc tgggctatgc ccgcgtcagc accgacgacc aggacttgac caaccaacgg
     1621 gccgaactgc acgcggccgg ctgcaccaag ctgttttccg agaagatcac cggcaccagg
     1681 cgcgaccgcc cggagctggc caggatgctt gaccacctac gccctggcga cgttgtgaca
     1741 gtgaccaggc tagaccgcct ggcccgcagc acccgcgacc tactggacat tgccgagcgc
     1801 atccaggagg ccggcgcggg cctgcgtagc ctggcagagc cgtgggccga caccaccacg
     1861 ccggccggcc gcatggtgtt gaccgtgttc gccggcattg ccgagttcga gcgttcccta
     1921 atcatcgacc gcacccggag cgggcgcgag gccgccaagg cccgaggcgt gaagtttggc
     1981 ccccgcccta ccctcacccc ggcacagatc gcgcacgccc gcgagctgat cgaccaggaa
     2041 ggccgcaccg tgaaagaggc ggctgcactg cttggcgtgc atcgctcgac cctgtaccgc
     2101 gcacttgagc gcagcgagga agtgacgccc accgaggcca ggcggcgcgg tgccttccgt
     2161 gaggacgcat tgaccgaggc cgacgccctg gcggccgccg agaatgaacg ccaagaggaa
     2221 caagcatgaa accgcaccag gacggccagg acgaaccgtt tttcattacc gaagagatcg
     2281 aggcggagat gatcgcggcc gggtacgtgt tcgagccgcc cgcgcacgtc tcaaccgtgc
     2341 ggctgcatga aatcctggcc ggtttgtctg atgccaagct ggcggcctgg ccggccagct
     2401 tggccgctga agaaaccgag cgccgccgtc taaaaaggtg atgtgtattt gagtaaaaca
     2461 gcttgcgtca tgcggtcgct gcgtatatga tgcgatgagt aaataaacaa atacgcaagg
     2521 ggaacgcatg aaggttatcg ctgtacttaa ccagaaaggc gggtcaggca agacgaccat
     2581 cgcaacccat ctagcccgcg ccctgcaact cgccggggcc gatgttctgt tagtcgattc
     2641 cgatccccag ggcagtgccc gcgattgggc ggccgtgcgg gaagatcaac cgctaaccgt
     2701 tgtcggcatc gaccgcccga cgattgaccg cgacgtgaag gccatcggcc ggcgcgactt
     2761 cgtagtgatc gacggagcgc cccaggcggc ggacttggct gtgtccgcga tcaaggcagc
     2821 cgacttcgtg ctgattccgg tgcagccaag cccttacgac atatgggcca ccgccgacct
     2881 ggtggagctg gttaagcagc gcattgaggt cacggatgga aggctacaag cggcctttgt
     2941 cgtgtcgcgg gcgatcaaag gcacgcgcat cggcggtgag gttgccgagg cgctggccgg
     3001 gtacgagctg cccattcttg agtcccgtat cacgcagcgc gtgagctacc caggcactgc
     3061 cgccgccggc acaaccgttc ttgaatcaga acccgagggc gacgctgccc gcgaggtcca
     3121 ggcgctggcc gctgaaatta aatcaaaact catttgagtt aatgaggtaa agagaaaatg
     3181 agcaaaagca caaacacgct aagtgccggc cgtccgagcg cacgcagcag caaggctgca
     3241 acgttggcca gcctggcaga cacgccagcc atgaagcggg tcaactttca gttgccggcg
     3301 gaggatcaca ccaagctgaa gatgtacgcg gtacgccaag gcaagaccat taccgagctg
     3361 ctatctgaat acatcgcgca gctaccagag taaatgagca aatgaataaa tgagtagatg
     3421 aattttagcg gctaaaggag gcggcatgga aaatcaagaa caaccaggca ccgacgccgt
     3481 ggaatgcccc atgtgtggag gaacgggcgg ttggccaggc gtaagcggct gggttgtctg
     3541 ccggccctgc aatggcactg gaacccccaa gcccgaggaa tcggcgtgac ggtcgcaaac
     3601 catccggccc ggtacaaatc ggcgcggcgc tgggtgatga cctggtggag aagttgaagg
     3661 ccgcgcaggc cgcccagcgg caacgcatcg aggcagaagc acgccccggt gaatcgtggc
     3721 aagcggccgc tgatcgaatc cgcaaagaat cccggcaacc gccggcagcc ggtgcgccgt
     3781 cgattaggaa gccgcccaag ggcgacgagc aaccagattt tttcgttccg atgctctatg
     3841 acgtgggcac ccgcgatagt cgcagcatca tggacgtggc cgttttccgt ctgtcgaagc
     3901 gtgaccgacg agctggcgag gtgatccgct acgagcttcc agacgggcac gtagaggttt
     3961 ccgcagggcc ggccggcatg gccagtgtgt gggattacga cctggtactg atggcggttt
     4021 cccatctaac cgaatccatg aaccgatacc gggaagggaa gggagacaag cccggccgcg
     4081 tgttccgtcc acacgttgcg gacgtactca agttctgccg gcgagccgat ggcggaaagc
     4141 agaaagacga cctggtagaa acctgcattc ggttaaacac cacgcacgtt gccatgcagc
     4201 gtacgaagaa ggccaagaac ggccgcctgg tgacggtatc cgagggtgaa gccttgatta
     4261 gccgctacaa gatcgtaaag agcgaaaccg ggcggccgga gtacatcgag atcgagctag
     4321 ctgattggat gtaccgcgag atcacagaag gcaagaaccc ggacgtgctg acggttcacc
     4381 ccgattactt tttgatcgat cccggcatcg gccgttttct ctaccgcctg gcacgccgcg
     4441 ccgcaggcaa ggcagaagcc agatggttgt tcaagacgat ctacgaacgc agtggcagcg
     4501 ccggagagtt caagaagttc tgtttcaccg tgcgcaagct gatcgggtca aatgacctgc
     4561 cggagtacga tttgaaggag gaggcggggc aggctggccc gatcctagtc atgcgctacc
     4621 gcaacctgat cgagggcgaa gcatccgccg gttcctaatg tacggagcag atgctagggc
     4681 aaattgccct agcaggggaa aaaggtcgaa aaggtctctt tcctgtggat agcacgtaca
     4741 ttgggaaccc aaagccgtac attgggaacc ggaacccgta cattgggaac ccaaagccgt
     4801 acattgggaa ccggtcacac atgtaagtga ctgatataaa agagaaaaaa ggcgattttt
     4861 ccgcctaaaa ctctttaaaa cttattaaaa ctcttaaaac ccgcctggcc tgtgcataac
     4921 tgtctggcca gcgcacagcc gaagagctgc aaaaagcgcc tacccttcgg tcgctgcgct
     4981 ccctacgccc cgccgcttcg cgtcggccta tcgcggccgc tggccgctca aaaatggctg
     5041 gcctacggcc aggcaatcta ccagggcgcg gacaagccgc gccgtcgcca ctcgaccgcc
     5101 ggcgcccaca tcaaggcacc ctgcctcgcg cgtttcggtg atgacggtga aaacctctga
     5161 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     5221 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca
     5281 cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag attgtactga
     5341 gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
     5401 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5461 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     5521 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5581 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5641 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5701 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5761 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     5821 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     5881 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     5941 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     6001 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     6061 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6121 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6181 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6241 ttttggtcat gcattctagg tactaaaaca attcatccag taaaatataa tattttattt
     6301 tctcccaatc aggcttgatc cccagtaagt caaaaaatag ctcgacatac tgttcttccc
     6361 cgatatcctc cctgatcgac cggacgcaga aggcaatgtc ataccacttg tccgccctgc
     6421 cgcttctccc aagatcaata aagccactta ctttgccatc tttcacaaag atgttgctgt
     6481 ctcccaggtc gccgtgggaa aagacaagtt cctcttcggg cttttccgtc tttaaaaaat
     6541 catacagctc gcgcggatct ttaaatggag tgtcttcttc ccagttttcg caatccacat
     6601 cggccagatc gttattcagt aagtaatcca attcggctaa gcggctgtct aagctattcg
     6661 tatagggaca atccgatatg tcgatggagt gaaagagcct gatgcactcc gcatacagct
     6721 cgataatctt ttcagggctt tgttcatctt catactcttc cgagcaaagg acgccatcgg
     6781 cctcactcat gagcagattg ctccagccat catgccgttc aaagtgcagg acctttggaa
     6841 caggcagctt tccttccagc catagcatca tgtccttttc ccgttccaca tcataggtgg
     6901 tccctttata ccggctgtcc gtcattttta aatataggtt ttcattttct cccaccagct
     6961 tatatacctt agcaggagac attccttccg tatcttttac gcagcggtat ttttcgatca
     7021 gttttttcaa ttccggtgat attctcattt tagccattta ttatttcctt cctcttttct
     7081 acagtattta aagatacccc aagaagctaa ttataacaag acgaactcca attcactgtt
     7141 ccttgcattc taaaacctta aataccagaa aacagctttt tcaaagttgt tttcaaagtt
     7201 ggcgtataac atagtatcga cggagccgat tttgaaaccg cggtgatcac aggcagcaac
     7261 gctctgtcat cgttacaatc aacatgctac cctccgcgag atcatccgtg tttcaaaccc
     7321 ggcagcttag ttgccgttct tccgaatagc atcggtaaca tgagcaaagt ctgccgcctt
     7381 acaacggctc tcccgctgac gccgtcccgg actgatgggc tgcctgtatc gagtggtgat
     7441 tttgtgccga gctgccggtc ggggagctgt tggctggctg g
//
