LOCUS       sequence-60745-e        5580 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a luciferase gene disrupted by a QQR ZFN target site and a
            frame-shift mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5580)
  TITLE     Plasmids to assay for nuclease cleavage in planta
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5580)
  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            "Unpublished"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-qqr::LUC
FEATURES             Location/Qualifiers
     source          1..5580
                     /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             1159..2808
                     /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(1222..1240)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     polyA_signal    2878..3054
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     misc_feature    3062..3084
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA 
                     (truncated)"
     rep_origin      complement(3151..3739)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3231..3250)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3910..4725)
                     /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
                     KPDAPELFLKHGKGSVANVVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4633..4652
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4717..4736
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5016..5451
                     /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 gtctagactt cttcccctca
     1141 ggaggggaag aactcgagga agacgccaaa aacataaaga aaggcccggc gccattctat
     1201 ccgctggaag atggaaccgc tggagagcaa ctgcataagg ctatgaagag atacgccctg
     1261 gttcctggaa caattgcttt tacagatgca catatcgagg tggacatcac ttacgctgag
     1321 tacttcgaaa tgtccgttcg gttggcagaa gctatgaaac gatatgggct gaatacaaat
     1381 cacagaatcg tcgtatgcag tgaaaactct cttcaattct ttatgccggt gttgggcgcg
     1441 ttatttatcg gagttgcagt tgcgcccgcg aacgacattt ataatgaacg tgaattgctc
     1501 aacagtatgg gcatttcgca gcctaccgtg gtgttcgttt ccaaaaaggg gttgcaaaaa
     1561 attttgaacg tgcaaaaaaa gctcccaatc atccaaaaaa ttattatcat ggattctaaa
     1621 acggattacc agggatttca gtcgatgtac acgttcgtca catctcatct acctcccggt
     1681 tttaatgaat acgattttgt gccagagtcc ttcgataggg acaagacaat tgcactgatc
     1741 atgaactcct ctggatctac tggtctgcct aaaggtgtcg ctctgcctca tagaactgcc
     1801 tgcgtgagat tctcgcatgc cagagatcct atttttggca atcaaatcat tccggatact
     1861 gcgattttaa gtgttgttcc attccatcac ggttttggaa tgtttactac actcggatat
     1921 ttgatatgtg gatttcgagt cgtcttaatg tatagatttg aagaagagct gtttctgagg
     1981 agccttcagg attacaagat tcaaagtgcg ctgctggtgc caaccctatt ctccttcttc
     2041 gccaaaagca ctctgattga caaatacgat ttatctaatt tacacgaaat tgcttctggt
     2101 ggcgctcccc tctctaagga agtcggggaa gcggttgcca agaggttcca tctgccaggt
     2161 atcaggcaag gatatgggct cactgagact acatcagcta ttctgattac acccgagggg
     2221 gatgataaac cgggcgcggt cggtaaagtt gttccatttt ttgaagcgaa ggttgtggat
     2281 ctggataccg ggaaaacgct gggcgttaat caaagaggcg aactgtgtgt gagaggtcct
     2341 atgattatgt ccggttatgt aaacaatccg gaagcgacca acgccttgat tgacaaggat
     2401 ggatggctac attctggaga catagcttac tgggacgaag acgaacactt cttcatcgtt
     2461 gaccgcctga agtctctgat taagtacaaa ggctatcagg tggctcccgc tgaattggaa
     2521 tccatcttgc tccaacaccc caacatcttc gacgcaggtg tcgcaggtct tcccgacgat
     2581 gacgccggtg aacttcccgc cgccgttgtt gttttggagc acggaaagac gatgacggaa
     2641 aaagagatcg tggattacgt cgccagtcaa gtaacaaccg cgaaaaagtt gcgcggagga
     2701 gttgtgtttg tggacgaagt accgaaaggt cttaccggaa aactcgacgc aagaaaaatc
     2761 agagagatcc tcataaaggc caagaagggc ggaaagatcg ccgtgtaatt ctagctagag
     2821 aattaattcg gtacgctgaa atcaccagtc tctctctaca aatctatctc tctctatttt
     2881 ctccataaat aatgtgtgag tagtttcccg ataagggaaa ttagggttct tatagggttt
     2941 cgctcatgtg ttgagcatat aagaaaccct tagtatgtat ttgtatttgt aaaatacttc
     3001 tatcaataaa atttctaatt cctaaaacca aaatccagta ctaaaatcca gatcgataac
     3061 gttacaccac aatatatcct gccaagatct catgtgagca aaaggccagc aaaaggccag
     3121 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     3181 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     3241 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     3301 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     3361 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     3421 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     3481 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     3541 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     3601 tggtatctgc gctctgctga agccagttac cttcggaaga agagttggta gctcttgatc
     3661 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     3721 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     3781 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     3841 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg tgtaacattg
     3901 gtctagtgat tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
     3961 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
     4021 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
     4081 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
     4141 agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc
     4201 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
     4261 tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac
     4321 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     4381 atcaggatat tcttctaata cctggaatgc tgttttccct gggatcgcag tggtgagtaa
     4441 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     4501 cagccagttt agtctgacca tctcatctgt aacaacattg gcaacgctac ctttgccatg
     4561 tttcagaaac aactctggcg catcgggctt cccatacaat cggtagattg tcgcacctga
     4621 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     4681 taatcgcggc cttgagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt
     4741 actgtttatg taagcagaca gttttattgt tcatgatgat atatttttat cttgtgcaat
     4801 gtaacatcag agattttgag acacaacgtg gctttgttga ataaatcgaa cttttgctga
     4861 gttgaaggat cagatcacgc atcttcccga caacgcagac cgttccgtgg caaagcaaaa
     4921 gttcaaaatc accaactggt ccacctacaa caaagctctc atcaaccgtg gctccctcac
     4981 tttctggctg gatgatgggg cgattcaggc gatccccatc caacagcccg ccgtcgagcg
     5041 ggctttttta tccccggaag cctgtggata gagggtagtt atccacgtga aaccgctaat
     5101 gccccgcaaa gccttgattc acggggcttt ccggcccgct ccaaaaacta tccacgtgaa
     5161 atcgctaatc agggtacgtg aaatcgctaa tcggagtacg tgaaatcgct aataaggtca
     5221 cgtgaaatcg ctaatcaaaa aggcacgtga gaacgctaat agccctttca gatcaacagc
     5281 ttgcaaacac ccctcgctcc ggcaagtagt tacagcaagt agtatgttca attagctttt
     5341 caattatgaa tatatatatc aattattggt cgcccttggc ttgtggacaa tgcgctacgc
     5401 gcaccggctc cgcccgtgga caaccgcaag cggttgccca ccgtcgagcg ccagcgcctt
     5461 tgcccacaac ccggcggccg gccgcaacag atcgttttat aaattttttt ttttgaaaaa
     5521 gaaaaagccc gaaaggcggc aacctctcgg gcttctggat ttccgatccc cggaattaga
//
