LOCUS       sequence-490172-        5780 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  35Sp::eGFP:35St cassette in L1 vector backbone. Expresses a nuclear 
            localized eGFP protein for transient plant transformation..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5780)
  AUTHORS   Abate BA, Hahn F, Chirivi D, Betti C, Fornara F, Molloy MC, Creasey 
            Krainer KM
  TITLE     Developing a Molecular Toolkit to ENABLE all to apply 
            CRISPR/Cas9-based Gene Editing in planta
  JOURNAL   bioRxiv 2025.11.09.687425
REFERENCE   2  (bases 1 to 5780)
  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: "bioRxiv 
            2025.11.09.687425"
COMMENT     Sequence Label: pGMF6
FEATURES             Location/Qualifiers
     source          1..5780
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     183..202
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     396..418
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     410..427
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     411..427
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    586..610
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     promoter        1708..2053
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     1996..2016
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             join(2144..2146,2147..2149,2150..2860)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2144..2146,2147..2149,2150..2860)
                     /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(2189..2210)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2450..2469)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2797..2818
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3165..3339
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     misc_feature    3384..3408
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     primer_bind     complement(3559..3575)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3559..3575)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3572..3594)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3583..3599
                     /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(3607..3613,3614..3631,3632..3637))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3652..3673
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3790..3807)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3961..4549)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4041..4060)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4720..5511,5512..5580))
                     /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     5343..5362
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5581..5685)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5753..5771
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgcacga ggccctttca tctcgcgcgt ttcggtgatg acggtgaaaa
       61 cctctgacac atgcagctcc cctagacggt cacagcttgt ctgtaagcgg atgccgggag
      121 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta
      181 tgcggcatca gagcagattg tactgagagt gcaccaaatg cggtgtgaaa taccgcacag
      241 atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg
      301 ggaagggcga tcggtgcggg cctcatcgct attacgccag ctggcgaaag ggggatgtgc
      361 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
      421 ggccagtgca acgcgatgac gatggatagc gattcatcga tgagctgacc cgatcgccgc
      481 cgccggaggg ttgcgtttga gacaggcgac agatactgat gggctgcctg tatcgagtgg
      541 tgattttgtg ccgagctgcc ggtcggggag ctgttggctg gctggtggca ggatatattg
      601 tggtgtaaac aaattgacgc ttagacaact taataacaca ttgcggacgt ttttaatgta
      661 ctggggtgga tgcagtgggc cccactctgt gaagacaatg ccgaattcgg atccggagga
      721 attccaatcc cacaaaaatc tgagcttaac agcacagttg ctcctctcag agcagaatcg
      781 ggtattcaac accctcatat caactactac gttgtgtata acggtccaca tgccggtata
      841 tacgatgact ggggttgtac aaaggcggca acaaacggcg ttcccggagt tgcacacaag
      901 aaatttgcca ctattacaga ggcaagagca gcagctgacg cgtacacaac aagtcagcaa
      961 acagacaggt tgaacttcat ccccaaagga gaagctcaac tcaagcccaa gagctttgct
     1021 aaggccctaa caagcccacc aaagcaaaaa gcccactggc tcacgctagg aaccaaaagg
     1081 cccagcagtg atccagcccc aaaagagatc tcctttgccc cggagattac aatggacgat
     1141 ttcctctatc tttacgatct aggaaggaag ttcgaaggtg aaggtgacga cactatgttc
     1201 accactgata atgagaaggt tagcctcttc aatttcagaa agaatgctga cccacagatg
     1261 gttagagagg cctacgcagc aagtctcatc aagacgatct acccgagtaa caatctccag
     1321 gagatcaaat accttcccaa gaaggttaaa gatgcagtca aaagattcag gactaattgc
     1381 atcaagaaca cagagaaaga catatttctc aagatcagaa gtactattcc agtatggacg
     1441 attcaaggct tgcttcataa accaaggcaa gtaatagaga ttggagtctc taaaaaggta
     1501 gttcctactg aatctaaggc catgcatgga gtctaagatt caaatcgagg atctaacaga
     1561 actcgccgtc aagactggcg aacagttcat acagagtctt ttacgactca atgacaagaa
     1621 gaaaatcttc gtcaacatgg tggagcacga cactctggtc tactccaaaa atgtcaaaga
     1681 tacagtctca gaagatcaaa gggctattga gacttttcaa caaaggataa tttcgggaaa
     1741 cctcctcgga ttccattgcc cagctatctg tcacttcatc gaaaggacag tagaaaagga
     1801 aggtggctcc tacaaatgcc atcattgcga taaaggaaag gctatcattc aagatctctc
     1861 tgccgacagt ggtcccaaag atggaccccc acccacgagg agcatcgtgg aaaaagaaga
     1921 ggttccaacc acgtctacaa agcaagtgga ttgatgtgac atctccactg acgtaaggga
     1981 tgacgcacaa tcccactatc cttcgcaaga cccttcctct atataaggaa gttcatttca
     2041 tttggagagg acacgctcga gtataagagc tcatttttac aacaattacc aacaacaaca
     2101 aacaacaaac aacattacaa ttacatttac aattatcgat acaatggtga gcaagggcga
     2161 ggagctgttc accggcgtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     2221 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
     2281 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
     2341 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     2401 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
     2461 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     2521 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
     2581 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt
     2641 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
     2701 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
     2761 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     2821 cgccgccggg atcactctcg gcatggacga gctgtacaag ggaggtgccg gcagtggcgc
     2881 tggcggagaa tttaagcgcg aagaacaagc ccgcaaggct aaagttaata acgaaaaaaa
     2941 gaccgaaatc gtcaagccag agtcttgctc caatgagggc gatgtgaagg atcttaaaag
     3001 gaaagactct gaggatggaa atgaagggga ggaagaagag gcttcgagta agcccaagaa
     3061 accgaaggtc gcactgagtc acttgcagga catagatgat acggaagctg accaagaaga
     3121 agaataatga gcttctctag ctagagtcga tcgacaagct cgagtttctc cataataatg
     3181 tgtgagtagt tcccagataa gggaattagg gttcctatag ggtttcgctc atgtgttgag
     3241 catataagaa acccttagta tgtatttgta tttgtaaaat acttctatca ataaaatttc
     3301 taattcctaa aaccaaaatc cagtactaaa atccagatcg ctgcaattgt cttctgcacg
     3361 aagtggttta aactatcagt gtttgacagg atatattggc gggtaaacct aagagaaaag
     3421 agcgtttatt agaataatcg gatatttaaa agggcgtgaa aaggtttatc cgttcgtcca
     3481 tttgtatgtg catgccaacc acagggttcc atcagttctg gaccagcgag ctgtgctgcg
     3541 actcgtggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     3601 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     3661 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     3721 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     3781 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     3841 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     3901 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     3961 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     4021 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     4081 ctctcctgtt ccgaccctgt cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     4141 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     4201 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     4261 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     4321 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     4381 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     4441 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     4501 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     4561 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     4621 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     4681 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     4741 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     4801 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     4861 agatccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     4921 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     4981 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     5041 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     5101 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     5161 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     5221 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     5281 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     5341 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     5401 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     5461 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     5521 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     5581 actctacctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     5641 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     5701 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     5761 tatcacgagg ccctttcatc
//
