LOCUS       sequence-472941-        5617 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a luciferase gene disrupted by an AtCRU3 TALEN Target 494 
            site and a frame-shift mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5617)
  TITLE     Plasmids to assay for nuclease cleavage in planta
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5617)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pGreenII Tfs-494::LUC
FEATURES             Location/Qualifiers
     source          1..5617
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    30..54
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     primer_bind     159..176
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    293..314
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(329..334,335..352,353..359)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    367..383
                     /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     372..394
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     391..407
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     391..407
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     426..446
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        428..446
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(500..519)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(501..519)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(529..546)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(529..545)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(538..560)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        763..1108
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     1051..1071
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             1204..2853
                     /codon_start=1
                     /gene="luc+"
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="enhanced luc+ version of the luciferase gene"
                     /translation="EDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAH
                     IEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPA
                     NDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSM
                     YTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHAR
                     DPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKI
                     QSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYG
                     LTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSG
                     YVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILL
                     QHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVF
                     VDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     primer_bind     complement(1267..1285)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     polyA_signal    2923..3099
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     misc_feature    3107..3129
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA 
                     (truncated)"
     rep_origin      complement(3196..3784)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3276..3295)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3955..4770)
                     /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(3955..4770)
                     /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     4678..4697
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4762..4781
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5061..5496
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
ORIGIN
        1 gatctcaaac aaacacatac agcgacttag tttacccgcc aatatatcct gtcaaggcct
       61 tcatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
      121 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
      181 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
      241 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
      301 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
      361 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
      421 agcgcgcaat taaccctcac taaagggaac aaaagctgga gctccaccgc ggtggcggcc
      481 gctctaggta cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt
      541 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
      601 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
      661 gttgcgcagc ctgaatggcg aatggaaatt gtaagcgtta atgttatcgt acccctactc
      721 caaaaatgtc aaagatacag tctcagaaga ccaaagggct attgagactt ttcaacaaag
      781 ggtaatttcg ggaaacctcc tcggattcca ttgcccagct atctgtcact tcatcgaaag
      841 gacagtagaa aaggaaggtg gctcctacaa atgccatcat tgcgataaag gaaaggctat
      901 cattcaagat gcctctgccg acagtggtcc caaagatgga cccccaccca cgaggagcat
      961 cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa gtggattgat gtgacatctc
     1021 cactgacgta agggatgacg cacaatccca ctatccttcg caagaccctt cctctatata
     1081 aggaagttca tttcatttgg agaggacagc ccaagctcat gtctagacct gcaaggacaa
     1141 caacaaggcc aaccatggca aggacgacag ggacaacaag gccaaccatg ggaaggactc
     1201 gaggaagacg ccaaaaacat aaagaaaggc ccggcgccat tctatccgct ggaagatgga
     1261 accgctggag agcaactgca taaggctatg aagagatacg ccctggttcc tggaacaatt
     1321 gcttttacag atgcacatat cgaggtggac atcacttacg ctgagtactt cgaaatgtcc
     1381 gttcggttgg cagaagctat gaaacgatat gggctgaata caaatcacag aatcgtcgta
     1441 tgcagtgaaa actctcttca attctttatg ccggtgttgg gcgcgttatt tatcggagtt
     1501 gcagttgcgc ccgcgaacga catttataat gaacgtgaat tgctcaacag tatgggcatt
     1561 tcgcagccta ccgtggtgtt cgtttccaaa aaggggttgc aaaaaatttt gaacgtgcaa
     1621 aaaaagctcc caatcatcca aaaaattatt atcatggatt ctaaaacgga ttaccaggga
     1681 tttcagtcga tgtacacgtt cgtcacatct catctacctc ccggttttaa tgaatacgat
     1741 tttgtgccag agtccttcga tagggacaag acaattgcac tgatcatgaa ctcctctgga
     1801 tctactggtc tgcctaaagg tgtcgctctg cctcatagaa ctgcctgcgt gagattctcg
     1861 catgccagag atcctatttt tggcaatcaa atcattccgg atactgcgat tttaagtgtt
     1921 gttccattcc atcacggttt tggaatgttt actacactcg gatatttgat atgtggattt
     1981 cgagtcgtct taatgtatag atttgaagaa gagctgtttc tgaggagcct tcaggattac
     2041 aagattcaaa gtgcgctgct ggtgccaacc ctattctcct tcttcgccaa aagcactctg
     2101 attgacaaat acgatttatc taatttacac gaaattgctt ctggtggcgc tcccctctct
     2161 aaggaagtcg gggaagcggt tgccaagagg ttccatctgc caggtatcag gcaaggatat
     2221 gggctcactg agactacatc agctattctg attacacccg agggggatga taaaccgggc
     2281 gcggtcggta aagttgttcc attttttgaa gcgaaggttg tggatctgga taccgggaaa
     2341 acgctgggcg ttaatcaaag aggcgaactg tgtgtgagag gtcctatgat tatgtccggt
     2401 tatgtaaaca atccggaagc gaccaacgcc ttgattgaca aggatggatg gctacattct
     2461 ggagacatag cttactggga cgaagacgaa cacttcttca tcgttgaccg cctgaagtct
     2521 ctgattaagt acaaaggcta tcaggtggct cccgctgaat tggaatccat cttgctccaa
     2581 caccccaaca tcttcgacgc aggtgtcgca ggtcttcccg acgatgacgc cggtgaactt
     2641 cccgccgccg ttgttgtttt ggagcacgga aagacgatga cggaaaaaga gatcgtggat
     2701 tacgtcgcca gtcaagtaac aaccgcgaaa aagttgcgcg gaggagttgt gtttgtggac
     2761 gaagtaccga aaggtcttac cggaaaactc gacgcaagaa aaatcagaga gatcctcata
     2821 aaggccaaga agggcggaaa gatcgccgtg taattctagc tagagaatta attcggtacg
     2881 ctgaaatcac cagtctctct ctacaaatct atctctctct attttctcca taaataatgt
     2941 gtgagtagtt tcccgataag ggaaattagg gttcttatag ggtttcgctc atgtgttgag
     3001 catataagaa acccttagta tgtatttgta tttgtaaaat acttctatca ataaaatttc
     3061 taattcctaa aaccaaaatc cagtactaaa atccagatcg ataacgttac accacaatat
     3121 atcctgccaa gatctcatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     3181 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     3241 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     3301 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     3361 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     3421 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     3481 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     3541 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     3601 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     3661 gctgaagcca gttaccttcg gaagaagagt tggtagctct tgatccggca aacaaaccac
     3721 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     3781 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     3841 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     3901 aaaatgaagt tttaaatcaa tctaaagtat atatgtgtaa cattggtcta gtgattagaa
     3961 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     4021 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     4081 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     4141 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     4201 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     4261 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     4321 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     4381 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     4441 taatacctgg aatgctgttt tccctgggat cgcagtggtg agtaaccatg catcatcagg
     4501 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     4561 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     4621 tggcgcatcg ggcttcccat acaatcggta gattgtcgca cctgattgcc cgacattatc
     4681 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggccttga
     4741 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
     4801 agacagtttt attgttcatg atgatatatt tttatcttgt gcaatgtaac atcagagatt
     4861 ttgagacaca acgtggcttt gttgaataaa tcgaactttt gctgagttga aggatcagat
     4921 cacgcatctt cccgacaacg cagaccgttc cgtggcaaag caaaagttca aaatcaccaa
     4981 ctggtccacc tacaacaaag ctctcatcaa ccgtggctcc ctcactttct ggctggatga
     5041 tggggcgatt caggcgatcc ccatccaaca gcccgccgtc gagcgggctt ttttatcccc
     5101 ggaagcctgt ggatagaggg tagttatcca cgtgaaaccg ctaatgcccc gcaaagcctt
     5161 gattcacggg gctttccggc ccgctccaaa aactatccac gtgaaatcgc taatcagggt
     5221 acgtgaaatc gctaatcgga gtacgtgaaa tcgctaataa ggtcacgtga aatcgctaat
     5281 caaaaaggca cgtgagaacg ctaatagccc tttcagatca acagcttgca aacacccctc
     5341 gctccggcaa gtagttacag caagtagtat gttcaattag cttttcaatt atgaatatat
     5401 atatcaatta ttggtcgccc ttggcttgtg gacaatgcgc tacgcgcacc ggctccgccc
     5461 gtggacaacc gcaagcggtt gcccaccgtc gagcgccttt gcccacaacc cggcggccgg
     5521 ccgcaacaga tcgttttata aatttttttt tttgaaaaag aaaaagcccg aaaggcggca
     5581 acctctcggg cttctggatt tccgatcccc ggaatta
//
