LOCUS       sequence-322860-        5545 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pGREEN-based cloning construct for expression in planta.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5545)
  AUTHORS   Pratt AI, Knoblauch J, Kunz HH
  TITLE     An updated pGREEN-based vector suite for cost-effective cloning in 
            plant molecular biology.
  JOURNAL   MicroPubl Biol. 2020 Oct 7;2020. doi: 
            10.17912/micropub.biology.000317.
  PUBMED    33274321
REFERENCE   2  (bases 1 to 5545)
  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: "MicroPubl 
            Biol."; date: "2020-10-7"; volume: "2020. doi"; pages: " 
            10.17912/micropub.biology.000317"
COMMENT     Sequence Label: pG20_FLAG_Hyg
FEATURES             Location/Qualifiers
     source          1..5545
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(497..1312)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(497..1312)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1220..1239
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1304..1323
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1603..2038
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     misc_feature    2165..2187
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA 
                     (truncated)"
     polyA_signal    2195..2371
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     CDS             complement(2603..3628)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     promoter        complement(3648..3993)
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     complement(3685..3705)
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             4721..4744
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    5168..5192
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(join(5283..5545,1..326))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5363..5382)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
       61 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
      121 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
      181 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaagaaga gttggtagct
      241 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
      301 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
      361 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
      421 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgtgt
      481 aacattggtc tagtgattag aaaaactcat cgagcatcaa atgaaactgc aatttattca
      541 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
      601 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
      661 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
      721 caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga
      781 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
      841 tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat
      901 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
      961 cacctgaatc aggatattct tctaatacct ggaatgctgt tttccctggg atcgcagtgg
     1021 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     1081 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     1141 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcgg tagattgtcg
     1201 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     1261 tggaatttaa tcgcggcctt gagcaagacg tttcccgttg aatatggctc ataacacccc
     1321 ttgtattact gtttatgtaa gcagacagtt ttattgttca tgatgatata tttttatctt
     1381 gtgcaatgta acatcagaga ttttgagaca caacgtggct ttgttgaata aatcgaactt
     1441 ttgctgagtt gaaggatcag atcacgcatc ttcccgacaa cgcagaccgt tccgtggcaa
     1501 agcaaaagtt caaaatcacc aactggtcca cctacaacaa agctctcatc aaccgtggct
     1561 ccctcacttt ctggctggat gatggggcga ttcaggcgat ccccatccaa cagcccgccg
     1621 tcgagcgggc ttttttatcc ccggaagcct gtggatagag ggtagttatc cacgtgaaac
     1681 cgctaatgcc ccgcaaagcc ttgattcacg gggctttccg gcccgctcca aaaactatcc
     1741 acgtgaaatc gctaatcagg gtacgtgaaa tcgctaatcg gagtacgtga aatcgctaat
     1801 aaggtcacgt gaaatcgcta atcaaaaagg cacgtgagaa cgctaatagc cctttcagat
     1861 caacagcttg caaacacccc tcgctccggc aagtagttac agcaagtagt atgttcaatt
     1921 agcttttcaa ttatgaatat atatatcaat tattggtcgc ccttggcttg tggacaatgc
     1981 gctacgcgca ccggctccgc ccgtggacaa ccgcaagcgg ttgcccaccg tcgagcgcct
     2041 ttgcccacaa cccggcggcc ggccgcaaca gatcgtttta taaatttttt tttttgaaaa
     2101 agaaaaagcc cgaaaggcgg caacctctcg ggcttctgga tttccgatcc ccggaattag
     2161 atcttggcag gatatattgt ggtgtaacgt tatcgatctg gattttagta ctggattttg
     2221 gttttaggaa ttagaaattt tattgataga agtattttac aaatacaaat acatactaag
     2281 ggtttcttat atgctcaaca catgagcgaa accctataag aaccctaatt tcccttatcg
     2341 ggaaactact cacacattat ttatggagaa aatagagaga gatagatttg tagagagaga
     2401 ctggtgattt cagcgtaccg aattaattcc tgaatcactg cgaccggccc tcccgcgacc
     2461 cagccgagcg agcttagcga actgtggacg agaactgtgc caccaagcgt aaggccgttc
     2521 tctcgcattt gccttgctag gctcgcgcga gttgctggct gaggcgttct cgaaatcagc
     2581 tcttgttcgg tcggcatcta ctctattcct ttgccctcgg acgagtgctg gggcgtcggt
     2641 ttccactatc ggcgagtact tctacacagc catcggtcca gacggccgcg cttctgcggg
     2701 cgatttgtgt acgcccgaca gtcccggctc cggatcggac gattgcgtcg catcgaccct
     2761 gcgcccaagc tgcatcatcg aaattgccgt caaccaagct ctgatagagt tggtcaagac
     2821 caatgcggag catatacgcc cggagccttg gcgatcctgc aagctccgga tgcctccgct
     2881 cgaagtagcg cgtctgctgc tccatacaag ccaaccacgg cctccagaag aagatgttgg
     2941 cgacctcgta ttgggaatcc ccgaacatcg cctcgctcca gtcaatgacc gctgttatgc
     3001 ggccattgtc cgtcaggaca ttgttggagc cgaaatccgc gtgcacgagg tgccggactt
     3061 cggggcagtc ctcggcccaa agcatcagct catcgagagc ctgcgcgacg gacgcactga
     3121 cggtgtcgtc catcacagtt tgccagtgat acacatgggg atcagcaatc gcgcatatga
     3181 aatcacgcca tgtagtgtat tgaccgattc cttgcggtcc gaatgggccg aacccgctcg
     3241 tctggctaag atcggccgca gcgatagcat ccatagcctc cgcgaccggc tgaagaacag
     3301 cgggcagttc ggtttcaggc aggtcttgca acgtgacacc ctgtgcacgg cgggagatgc
     3361 aataggtcag gctctcgctg aactccccaa tgtcaagcac ttccggaatc gggagcgcgg
     3421 ccgatgcaaa gtgccgataa acataacgat ctttgtagaa accatcggcg cagctattta
     3481 cccgcaggac atatccacgc cctcctacat cgaagctgaa agcacgagat tcttcgccct
     3541 ccgagagctg catcaggtcg gagacgctgt cgaacttttc gatcagaaac ttctcgacag
     3601 acgtcgcggt gagttcaggc tttttcatag gggggatcag cttgggctgt cctctccaaa
     3661 tgaaatgaac ttccttatat agaggaaggg tcttgcgaag gatagtggga ttgtgcgtca
     3721 tcccttacgt cagtggagat gtcacatcaa tccacttgct ttgaagtcgt ggttggaacg
     3781 acttcttttt ccacgatgct cctcgtgggt gggggtccat ctttgggacc actgtcggca
     3841 gaggcatctt gaatgatagc ctttccttta tcgcaatgat ggcatttgta ggagccacct
     3901 tccttttcta ctgtcctttc gatgaagtga cagatagctg ggcaatggaa tccgaggagg
     3961 tttcccgaaa ttaccctttg ttgaaaagtc tcaataaagc ttcgacgagt cagtaataaa
     4021 cggcgtcaaa gtggttgcag ccggcacaca cgagtcgtgt ttatcaactc aaagcacaaa
     4081 tacttttcct caacctaaaa ataaggcaat tagccaaaaa caactttgcg tgtaaacaac
     4141 gctcaataca cgtgtcattt tattattagc tattgcttca ccgccttagc tttctcgtga
     4201 cctagtcgtc ctcgtctttt cttcttcttc ttctataaaa caatacccaa agacctcttc
     4261 ttcttcacaa ttcagatttc aatttctcaa aatcttaaaa actttctctc aattctctct
     4321 accgtgatca aggtaaattt ctgtgttcct tattctctca aaatcttcga ttttgttttc
     4381 gttcgatccc aatttcgtat atgttctttg gtttagattc tgttaatctt agatcgaaga
     4441 cgattttctg ggtttgatcg ttagatatca tcttaattct cgattagggt ttcatagata
     4501 tcatccgatt tgttcaaata atttgagttt tgtcgaataa ttactcttcg atttgtgatt
     4561 tctatctaga tctggtgtta gtttctagtt tgtgcgatcg aatttgtcga ttaatctgag
     4621 tttttctgat taacagacta gagttaaccg ggctcaggcc tggcgcgcca ctagtggatc
     4681 catcgatagt actgtcgacc tcgagggtac cgctcccggg gactacaagg atgacgatga
     4741 caagtaagag ctcatatgaa gatgaagatg aaatatttgg tgtgtcaaat aaatagcttg
     4801 tgtgcttaag tttgtgtttt tttcttggct tgttgtgtta tgaatttgtg gctttttcta
     4861 atattaaatg aatgtaagat ctcattataa tgaataaaca aatgtttcta taatccattg
     4921 tgaatgtttt gttggatctc ttctgcagca tataactact gtatgtgcta tggtatggac
     4981 tatggaatat gattaaagat aaggaattca gatcggctga gtggctcctt caacgttgcg
     5041 gttctgtcag ttccaaacgt aaaacggctt gtcccgcgtc atcggcgggg gtcataacgt
     5101 gactccctta attctccgct catgatcaga ttgtcgtttc ccgccttcag tttaaactat
     5161 cagtgtttga caggatatat tggcgggtaa actaagtcgc tgtatgtgtt tgtttgagat
     5221 ctcatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     5281 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     5341 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     5401 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     5461 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     5521 tccaagctgg gctgtgtgca cgaac
//
