LOCUS       sequence-312954-        8790 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 1 Position 3 Golden Gate vector, 
            pL1M-R3-UbqP-Loxp-ER-Targ-mCherry-HDEL-Loxp-eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8790)
  AUTHORS   Harrington SA, Backhaus AE, Fox S, Rogers C, Borrill P, Uauy C, 
            Richardson A
  TITLE     A heat-shock inducible system for flexible gene expression in 
            cereals.
  JOURNAL   Plant Methods. 2020 Oct 14;16:137. doi: 10.1186/s13007-020-00677-3. 
            eCollection 2020.
  PUBMED    33072173
REFERENCE   2  (bases 1 to 8790)
  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: "Plant 
            Methods."; date: "2020-10-14"; volume: "16:137. doi"; pages: " 
            10.1186/s13007-020-00677-3. eCollection 2020"
COMMENT     Sequence Label: EC71167
FEATURES             Location/Qualifiers
     source          1..8790
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    100..124
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     promoter        170..2166
                     /label=Ubi promoter
                     /note="maize polyubiquitin gene promoter"
     protein_bind    complement(2177..2210)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2295..2999
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(2437..2455)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    3055..3229
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     protein_bind    complement(3239..3272)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             join(3357..3359,3360..3362,3363..4073)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3357..3359,3360..3362,3363..4073)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3402..3423)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3663..3682)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4010..4031
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    4692..4716
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     rep_origin      4808..5439
                     /label=oriV
                     /note="incP origin of replication"
     promoter        5526..5630
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5631..5699,5700..6491)
                     /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(5849..5868)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6662..7250
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7151..7170
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             7586..8734
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
ORIGIN
        1 accctgtggt tggcatgcac atacaaatgg acgaacggat aaaccttttc acgccctttt
       61 aaatatccga ttattctaat aaacgctctt ttctcttagg tttacccgcc aatatatcct
      121 gtcaaacact gatagtttaa accacttcgt gcagaagaca agtaaggagc tgcagtgcag
      181 cgtgacccgg tcgtgcccct ctctagagat aatgagcatt gcatgtctaa gttataaaaa
      241 attaccacat attttttttg tcacacttgt ttgaagtgca gtttatctat ctttatacat
      301 atatttaaac tttactctac gaataatata atctatagta ctacaataat atcagtgttt
      361 tagagaatca tataaatgaa cagttagaca tggtctaaag gacaattgag tattttgaca
      421 acaggactct acagttttat ctttttagtg tgcatgtgtt ctcctttttt tttgcaaata
      481 gcttcaccta tataatactt catccatttt attagtacat ccatttaggg tttagggtta
      541 atggttttta tagactaatt tttttagtac atctatttta ttctatttta gcctctaaat
      601 taagaaaact aaaactctat tttagttttt ttatttaata atttagatat aaaatagaat
      661 aaaataaagt gactaaaaat taaacaaata ccctttaaga aattaaaaaa actaaggaaa
      721 catttttctt gtttcgagta gataatgcca gcctgttaaa cgccgtcgac gagtctaacg
      781 gacaccaacc agcgaaccag cagcgtcgcg tcgggccaag cgaagcagac ggcacggcat
      841 ctctgtcgct gcctctggac ccctctcgag agttccgctc caccgttgga cttgctccgc
      901 tgtcggcatc cagaaattgc gtggcggagc ggcagacgtg agccggcacg gcaggcggcc
      961 tcctcctcct ctcacggcac cggcagctac gggggattcc tttcccaccg ctccttcgct
     1021 ttcccttcct cgcccgccgt aataaataga caccccctcc acaccctctt tccccaacct
     1081 cgtgttgttc ggagcgcaca cacacacaac cagatctccc ccaaatccac ccgtcggcac
     1141 ctccgcttca aggtacgccg ctcgtcctcc cccccccccc ctctctacct tctctagatc
     1201 ggcgttccgg tccatggtta gggcccggta gttctacttc tgttcatgtt tgtgttagat
     1261 ccgtgtttgt gttagatccg tgctgctagc gttcgtacac ggatgcgacc tgtacgtcag
     1321 acacgttctg attgctaact tgccagtgtt tctctttggg gaatcctggg atggctctag
     1381 ccgttccgca gacgggatcg atttcatgat tttttttgtt tcgttgcata gggtttggtt
     1441 tgcccttttc ctttatttca atatatgccg tgcacttgtt tgtcgggtca tcttttcatg
     1501 cttttttttg tcttggttgt gatgatgtgg tctggttggg cggtcgttct agatcggagt
     1561 agaattaatt ctgtttcaaa ctacctggtg gatttattaa ttttggatct gtatgtgtgt
     1621 gccatacata ttcatagtta cgaattgaag atgatggatg gaaatatcga tctaggatag
     1681 gtatacatgt tgatgcgggt tttactgatg catatacaga gatgcttttt gttcgcttgg
     1741 ttgtgatgat gtggtgtggt tgggcggtcg ttcattcgtt ctagatcgga gtagaatact
     1801 gtttcaaact acctggtgta tttattaatt ttggaactgt atgtgtgtgt catacatctt
     1861 catagttacg agtttaagat ggatggaaat atcgatctag gataggtata catgttgatg
     1921 tgggttttac tgatgcatat acatgatggc atatgcagca tctattcata tgctctaacc
     1981 ttgagtacct atctattata ataaacaagt atgttttata attattttga tcttgatata
     2041 cttggatgat ggcatatgca gcagctatat gtggattttt ttagccctgc cttcatacgc
     2101 tatttatttg cttggtactg tttcttttgt cgatgctcac cctgttgttt ggtgttactt
     2161 ctgcagtact gacctaataa cttcgtatag catacattat acgaagttat attaagggtt
     2221 gaatgaaaac taatcttttt ctctttctca tcttttcact tctcctatca ttatcctcgg
     2281 ccgaattcgg aggtgtgagc aagggcgagg aggataacat ggccatcatc aaggagttca
     2341 tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg
     2401 agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg
     2461 gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct
     2521 acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca
     2581 agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct
     2641 ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg
     2701 acggccccgt aatgcagaag aaaaccatgg gctgggaggc ctcctccgag cggatgtacc
     2761 ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc
     2821 actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg
     2881 cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg
     2941 aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagg
     3001 gtggccatga tgagctttaa gcttctctag ctagagtcga tcgacaagct cgagtttctc
     3061 cataataatg tgtgagtagt tcccagataa gggaattagg gttcctatag ggtttcgctc
     3121 atgtgttgag catataagaa acccttagta tgtatttgta tttgtaaaat acttctatca
     3181 ataaaatttc taattcctaa aaccaaaatc cagtactaaa atccagatcg ctgacctaat
     3241 aacttcgtat agcatacatt atacgaagtt atattaaggg ttgaatgaaa actaatcttt
     3301 ttctctttct catcttttca cttctcctat cattatcctc ggccgaattc ggaggtatgg
     3361 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     3421 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     3481 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     3541 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     3601 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     3661 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     3721 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     3781 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     3841 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     3901 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     3961 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     4021 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagggtggcc
     4081 atgatgagct ttaagcttgc tctcaagatc aaaggcttaa aaagctgggg ttttatgaat
     4141 gggatcaaag tttctttttt tcttttatat ttgcttctcc atttgtttgt ttcatttccc
     4201 tttttgtttt cgtttctatg atgcacttgt gtgtgacaaa ctctctgggt ttttacttac
     4261 gtctgcgttt caaaaaaaaa aaccgctttc gttttgcgtt ttagtcccat tgttttgtag
     4321 ctctgagtga tcgaattgat gcctctttat tccttttgtt ccctataatt tctttcaaaa
     4381 ctcagaagaa aaaccttgaa actctttgca atgttaatat aagtattgta taagattttt
     4441 attgatttgg ttattagtct tacttttgct acctccatct tcacttggaa ctgatattct
     4501 gaatagttaa agcgttacat gtgttccatt cacaaatgaa cttaaactag cacaaagtca
     4561 gatattttaa gatcgcacca tttcgctgag ctcgaattct agtttgtctt cacagagtgg
     4621 ggcccactgc atccacccca gtacattaaa aacgtccgca atgtgttatt aagttgtcta
     4681 agcgtcaatt tgtttacacc acaatatatc ctgccaccag ccagccaaca gctccccgac
     4741 cggcagctcg gcacaaaatc accactcgat acaggcagcc catcagtcag atcaggatct
     4801 cctttgcgac gctcaccggg ctggttgccc tcgccgctgg gctggcggcc gtctatggcc
     4861 ctgcaaacgc gccagaaacg ccgtcgaagc cgtgtgcgag acaccgcggc cgccggcgtt
     4921 gtggatacct cgcggaaaac ttggccctca ctgacagatg aggggcggac gttgacactt
     4981 gaggggccga ctcacccggc gcggcgttga cagatgaggg gcaggctcga tttcggccgg
     5041 cgacgtggag ctggccagcc tcgcaaatcg gcgaaaacgc ctgattttac gcgagtttcc
     5101 cacagatgat gtggacaagc ctggggataa gtgccctgcg gtattgacac ttgaggggcg
     5161 cgactactga cagatgaggg gcgcgatcct tgacacttga ggggcagagt gctgacagat
     5221 gaggggcgca cctattgaca tttgaggggc tgtccacagg cagaaaatcc agcatttgca
     5281 agggtttccg cccgtttttc ggccaccgct aacctgtctt ttaacctgct tttaaaccaa
     5341 tatttataaa ccttgttttt aaccagggct gcgccctgtg cgcgtgaccg cgcacgccga
     5401 aggggggtgc ccccccttct cgaaccctcc cggcccgcta acgcgggcct cccatccccc
     5461 caggggctgc gcccctcggc cgcgaacggc ctcaccccaa aaatggcagc gctggccaat
     5521 tcgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     5581 tgagacaata accttgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     5641 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     5701 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     5761 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     5821 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     5881 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     5941 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     6001 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     6061 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     6121 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     6181 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     6241 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     6301 cggctggctg gtttattgct gataaatctg gagccggtga gcgtggttct cgcggtatca
     6361 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     6421 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     6481 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     6541 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     6601 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     6661 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     6721 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     6781 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     6841 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     6901 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     6961 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     7021 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     7081 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     7141 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     7201 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     7261 acgcggcctt tttacggttc ctggcagatc ctagatgtgg cgcaacgatg ccggcgacaa
     7321 gcaggagcgc accgacttct tccgcatcaa gtgttttggc tctcaggccg aggcccacgg
     7381 caagtatttg ggcaaggggt cgctggtatt cgtgcagggc aagattcgga ataccaagta
     7441 cgagaaggac ggccagacgg tctacgggac cgacttcatt gccgataagg tggattatct
     7501 ggacaccaag gcaccaggcg ggtcaaatca ggaataaggg cacattgccc cggcgtgagt
     7561 cggggcaatc ccgcaaggag ggtgaatgaa tcggacgttt gaccggaagg catacaggca
     7621 agaactgatc gacgcggggt tttccgccga ggatgccgaa accatcgcaa gccgcaccgt
     7681 catgcgtgcg ccccgcgaaa ccttccagtc cgtcggctcg atggtccagc aagctacggc
     7741 caagatcgag cgcgacagcg tgcaactggc tccccctgcc ctgcccgcgc catcggccgc
     7801 cgtggagcgt tcgcgtcgtc ttgaacagga ggcggcaggt ttggcgaagt cgatgaccat
     7861 cgacacgcga ggaactatga cgaccaagaa gcgaaaaacc gccggcgagg acctggcaaa
     7921 acaggtcagc gaggccaagc aggccgcgtt gctgaaacac acgaagcagc agatcaagga
     7981 aatgcagctt tccttgttcg atattgcgcc gtggccggac acgatgcgag cgatgccaaa
     8041 cgacacggcc cgctctgccc tgttcaccac gcgcaacaag aaaatcccgc gcgaggcgct
     8101 gcaaaacaag gtcattttcc acgtcaacaa ggacgtgaag atcacctaca ccggcgtcga
     8161 gctgcgggcc gacgatgacg aactggtgtg gcagcaggtg ttggagtacg cgaagcgcac
     8221 ccctatcggc gagccgatca ccttcacgtt ctacgagctt tgccaggacc tgggctggtc
     8281 gatcaatggc cggtattaca cgaaggccga ggaatgcctg tcgcgcctac aggcgacggc
     8341 gatgggcttc acgtccgacc gcgttgggca cctggaatcg gtgtcgctgc tgcaccgctt
     8401 ccgcgtcctg gaccgtggca agaaaacgtc ccgttgccag gtcctgatcg acgaggaaat
     8461 cgtcgtgctg tttgctggcg accactacac gaaattcata tgggagaagt accgcaagct
     8521 gtcgccgacg gcccgacgga tgttcgacta tttcagctcg caccgggagc cgtacccgct
     8581 caagctggaa accttccgcc tcatgtgcgg atcggattcc acccgcgtga agaagtggcg
     8641 cgagcaggtc ggcgaagcct gcgaagagtt gcgaggcagc ggcctggtgg aacacgcctg
     8701 ggtcaatgat gacctggtgc attgcaaacg ctagggcctt gtggggtcag ttccggctgg
     8761 gggttcagca gccagcgcct gatctgggga
//
