LOCUS       sequence-325557-        8414 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry clone (attL1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8414)
  AUTHORS   Ren Q, Sretenovic S, Liu S, Tang X, Huang L, He Y, Liu L, Guo Y, 
            Zhong Z, Liu G, Cheng Y, Zheng X, Pan C, Yin D, Zhang Y, Li W, Qi L,
            Li C, Qi Y, Zhang Y
  TITLE     PAM-less plant genome editing using a CRISPR-SpRY toolbox.
  JOURNAL   Nat Plants. 2021 Jan 4. pii: 10.1038/s41477-020-00827-4. doi: 
            10.1038/s41477-020-00827-4.
  PUBMED    33398158
REFERENCE   2  (bases 1 to 8414)
  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: "Nat Plants.
            2021 Jan 4. pii: 10.1038/s41477-020-00827-4. doi: 
            10.1038/s41477-020-00827-4."
COMMENT     Sequence Label: pYPQ262B-ABE8e
FEATURES             Location/Qualifiers
     source          1..8414
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..100
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             140..160
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             761..826
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             833..853
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     RBS             4891..4899
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             4979..5026
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     protein_bind    5701..5824
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(5841..5860)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5842..5860)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5865..5881)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5865..5881)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             6312..7103
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      7196..7784
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7685..7704
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7938..7955
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(8114..8141)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(8233..8319)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     8368..8390
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8382..8399
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8383..8399
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 caaataatga ttttattttg actgatagtg acctgttcgt tgcaacaaat tgatgagcaa
       61 tgctttttta taatgccaac tttgtacaaa aaagcaggct ccgatgaaac ggacagccga
      121 cggaagcgag ttcgagtcac caaagaagaa gcggaaagtc tctgaggtgg agttttccca
      181 cgagtactgg atgagacatg ccctgaccct ggccaagagg gcacgggatg agagggaggt
      241 gcctgtggga gccgtgctgg tgctgaacaa tagagtgatc ggcgagggct ggaacagagc
      301 catcggcctg cacgacccaa cagcccatgc cgaaattatg gccctgagac agggcggcct
      361 ggtcatgcag aactacagac tgattgacgc caccctgtac gtgacattcg agccttgcgt
      421 gatgtgcgcc ggcgccatga tccactctag gatcggccgc gtggtgtttg gcgtgaggaa
      481 ctcaaaaaga ggcgccgcag gctccctgat gaacgtgctg aactaccccg gcatgaatca
      541 ccgcgtcgaa attaccgagg gaatcctggc agatgaatgt gccgccctgc tgtgcgattt
      601 ctatcggatg cctagacagg tgttcaatgc tcagaagaag gcccagagct ccatcaactc
      661 cggaggatct agcggaggct cctctggctc tgagacacct ggcacaagcg agagcgcaac
      721 acctgaaagc agcgggggca gcagcggggg gtcaaccatg gattacaagg accacgacgg
      781 ggattacaag gaccacgaca ttgattacaa ggatgatgat gacaagatgg ctccgaagaa
      841 gaagaggaag gttggcatcc acggggtgcc agctgctgac aagaagtact cgatcggcct
      901 cgctattggg actaactctg ttggctgggc cgtgatcacc gacgagtaca aggtgccctc
      961 aaagaagttc aaggtcctgg gcaacaccga tcggcattcc atcaagaaga atctcattgg
     1021 cgctctcctg ttcgacagcg gcgagacggc tgagaggacg cggctcaagc gcaccgcccg
     1081 caggcggtac acgcgcagga agaatcgcat ctgctacctg caggagattt tctccaacga
     1141 gatggcgaag gttgacgatt ctttcttcca caggctggag gagtcattcc tcgtggagga
     1201 ggataagaag cacgagcggc atccaatctt cggcaacatt gtcgacgagg ttgcctacca
     1261 cgagaagtac cctacgatct accatctgcg gaagaagctc gtggactcca cagataaggc
     1321 ggacctccgc ctgatctacc tcgctctggc ccacatgatt aagttcaggg gccatttcct
     1381 gatcgagggg gatctcaacc cggacaatag cgatgttgac aagctgttca tccagctcgt
     1441 gcagacgtac aaccagctct tcgaggagaa ccccattaat gcgtcaggcg tcgacgcgaa
     1501 ggctatcctg tccgctaggc tctcgaagtc tcggcgcctc gagaacctga tcgcccagct
     1561 gccgggcgag aagaagaacg gcctgttcgg gaatctcatt gcgctcagcc tggggctcac
     1621 gcccaacttc aagtcgaatt tcgatctcgc tgaggacgcc aagctgcagc tctccaagga
     1681 cacatacgac gatgacctgg ataacctcct ggcccagatc ggcgatcagt acgcggacct
     1741 gttcctcgct gccaagaatc tgtcggacgc catcctcctg tctgatattc tcagggtgaa
     1801 caccgagatt acgaaggctc cgctctcagc ctccatgatc aagcgctacg acgagcacca
     1861 tcaggatctg accctcctga aggcgctggt caggcagcag ctccccgaga agtacaagga
     1921 gatcttcttc gatcagtcga agaacggcta cgctgggtac attgacggcg gggcctctca
     1981 ggaggagttc tacaagttca tcaagccgat tctggagaag atggacggca cggaggagct
     2041 gctggtgaag ctcaatcgcg aggacctcct gaggaagcag cggacattcg ataacggcag
     2101 catcccacac cagattcatc tcggggagct gcacgctatc ctgaggaggc aggaggactt
     2161 ctaccctttc ctcaaggata accgcgagaa gatcgagaag attctgactt tcaggatccc
     2221 gtactacgtc ggcccactcg ctaggggcaa ctcccgcttc gcttggatga cccgcaagtc
     2281 agaggagacg atcacgccgt ggaacttcga ggaggtggtc gacaagggcg ctagcgctca
     2341 gtcgttcatc gagaggatga cgaatttcga caagaacctg ccaaatgaga aggtgctccc
     2401 taagcactcg ctcctgtacg agtacttcac agtctacaac gagctgacta aggtgaagta
     2461 tgtgaccgag ggcatgagga agccggcttt cctgtctggg gagcagaaga aggccatcgt
     2521 ggacctcctg ttcaagacca accggaaggt cacggttaag cagctcaagg aggactactt
     2581 caagaagatt gagtgcttcg attcggtcga gatctctggc gttgaggacc gcttcaacgc
     2641 ctccctgggg acctaccacg atctcctgaa gatcattaag gataaggact tcctggacaa
     2701 cgaggagaat gaggatatcc tcgaggacat tgtgctgaca ctcactctgt tcgaggaccg
     2761 ggagatgatc gaggagcgcc tgaagactta cgcccatctc ttcgatgaca aggtcatgaa
     2821 gcagctcaag aggaggaggt acaccggctg ggggaggctg agcaggaagc tcatcaacgg
     2881 cattcgggac aagcagtccg ggaagacgat cctcgacttc ctgaagagcg atggcttcgc
     2941 gaaccgcaat ttcatgcagc tgattcacga tgacagcctc acattcaagg aggatatcca
     3001 gaaggctcag gtgagcggcc agggggactc gctgcacgag catatcgcga acctcgctgg
     3061 ctcgccagct atcaagaagg ggattctgca gaccgtgaag gttgtggacg agctggtgaa
     3121 ggtcatgggc aggcacaagc ctgagaacat cgtcattgag atggcccggg agaatcagac
     3181 cacgcagaag ggccagaaga actcacgcga gaggatgaag aggatcgagg agggcattaa
     3241 ggagctgggg tcccagatcc tcaaggagca cccggtggag aacacgcagc tgcagaatga
     3301 gaagctctac ctgtactacc tccagaatgg ccgcgatatg tatgtggacc aggagctgga
     3361 tattaacagg ctcagcgatt acgacgtcga tcatatcgtt ccacagtcat tcctgaagga
     3421 tgactccatt gacaacaagg tcctcaccag gtcggacaag aaccggggca agtctgataa
     3481 tgttccttca gaggaggtcg ttaagaagat gaagaactac tggcgccagc tcctgaatgc
     3541 caagctgatc acgcagcgga agttcgataa cctcacaaag gctgagaggg gcgggctctc
     3601 tgagctggac aaggcgggct tcatcaagag gcagctggtc gagacacggc agatcactaa
     3661 gcacgttgcg cagattctcg actcacggat gaacactaag tacgatgaga atgacaagct
     3721 gatccgcgag gtgaaggtca tcaccctgaa gtcaaagctc gtctccgact tcaggaagga
     3781 tttccagttc tacaaggttc gggagatcaa caattaccac catgcccatg acgcgtacct
     3841 gaacgcggtg gtcggcacag ctctgatcaa gaagtaccca aagctcgaga gcgagttcgt
     3901 gtacggggac tacaaggttt acgatgtgag gaagatgatc gccaagtcgg agcaggagat
     3961 tggcaaggct accgccaagt acttcttcta ctctaacatt atgaatttct tcaagacaga
     4021 gatcactctg gccaatggcg agatccggaa gcgccccctc atcgagacga acggcgagac
     4081 gggggagatc gtgtgggaca agggcaggga tttcgcgacc gtcaggaagg ttctctccat
     4141 gccacaagtg aatatcgtca agaagacaga ggtccagact ggcgggttct ctaaggagtc
     4201 aattaggcct aagcggaaca gcgacaagct catcgcccgc aagaaggact gggatccgaa
     4261 gaagtacggc gggttcctgt ggcccactgt ggcctactcg gtcctggttg tggcgaaggt
     4321 tgagaagggc aagtccaaga agctcaagag cgtgaaggag ctgctgggga tcacgattat
     4381 ggagcgctcc agcttcgaga agaacccgat cgatttcctg gaggcgaagg gctacaagga
     4441 ggtgaagaag gacctgatca ttaagctccc caagtactca ctcttcgagc tggagaacgg
     4501 caggaagcgg atgctggctt ccgctaagca gctgcagaag gggaacgagc tggctctgcc
     4561 gtccaagtat gtgaacttcc tctacctggc ctcccactac gagaagctca agggcagccc
     4621 cgaggacaac gagcagaagc agctgttcgt cgagcagcac aagcattacc tcgacgagat
     4681 cattgagcag atttccgagt tctccaagcg cgtgatcctg gccgacgcga atctggataa
     4741 ggtcctctcc gcgtacaaca agcaccgcga caagccaatc agggagcagg ctgagaatat
     4801 cattcatctc ttcaccctga cgaggctcgg cgcccctagg gctttcaagt acttcgacac
     4861 aactatcgat ccgaagcagt acaggagcac taaggaggtc ctggacgcga ccctcatcca
     4921 ccagtcgatt accggcctct acgagacgcg catcgacctg tctcagctcg ggggcgacaa
     4981 gcggccagcg gcgacgaaga aggcggggca ggcgaagaag aagaagtgag ctcagagctt
     5041 tcgttcgtat catcggtttc gacaacgttc gtcaagttca atgcatcagt ttcattgcgc
     5101 acacaccaga atcctactga gtttgagtat tatggcattg ggaaaactgt ttttcttgta
     5161 ccatttgttg tgcttgtaat ttactgtgtt ttttattcgg ttttcgctat cgaactgtga
     5221 aatggaaatg gatggagaag agttaatgaa tgatatggtc cttttgttca ttctcaaatt
     5281 aatattattt gttttttctc ttatttgttg tgtgttgaat ttgaaattat aagagatatg
     5341 caaacatttt gttttgagta aaaatgtgtc aaatcgtggc ctctaatgac cgaagttaat
     5401 atgaggagta aaacacttgt agttgtacca ttatgcttat tcactaggca acaaatatat
     5461 tttcagacct agaaaagctg caaatgttac tgaatacaag tatgtcctct tgtgttttag
     5521 acatttatga actttccttt atgtaatttt ccagaatcct tgtcagattc taatcattgc
     5581 tttataatta tagttatact catggatttg tagttgagta tgaaaatatt ttttaatgca
     5641 ttttatgact tgccaattgt tccggaactc tagataagct taccggaaag ggcgaattcg
     5701 caactttgta tacaaaagtt gaacgagaaa cgtaaaatga tataaatatc aatatattaa
     5761 attagatttt gcataaaaaa cagactacat aatactgtaa aacacaacat atccagtcac
     5821 tatgccatcc agctgatatc ccctatagtg agtcgtatta catggtcata gctgtttcct
     5881 ggcagctctg gcccgtgtct caaaatctct gatgttacat tgcacaagat aaaaatatat
     5941 catcatgcct cctctggacc agccaggaca gaaatgcctc gacttcgctg ctacccaagg
     6001 ttgccgggtg acgcacaccg tggaaacgga tgaaggcacg aacccagtgg acataagcct
     6061 gttcggttcg taagctgtaa tgcaagtagc gtatgcgctc acgcaactgg tccagaacct
     6121 tgaccgaacg cagcggtggt aacggcgcag tggcggtttt catggcttgt tatgactgtt
     6181 tttttggggt acagtctatg cctcgggcat ccaagcagca agcgcgttac gccgtgggtc
     6241 gatgtttgat gttatggagc agcaacgatg ttacgcagca gggcagtcgc cctaaaacaa
     6301 agttaaacat tatgagggaa gcggtgatcg ccgaagtatc gactcaacta tcagaggtag
     6361 ttggcgtcat cgagcgccat ctcgaaccga cgttgctggc cgtacatttg tacggctccg
     6421 cagtggatgg cggcctgaag ccacacagtg atattgattt gctggttacg gtgaccgtaa
     6481 ggcttgatga aacaacgcgg cgagctttga tcaacgacct tttggaaact tcggcttccc
     6541 ctggagagag cgagattctc cgcgctgtag aagtcaccat tgttgtgcac gacgacatca
     6601 ttccgtggcg ttatccagct aagcgcgaac tgcaatttgg agaatggcag cgcaatgaca
     6661 ttcttgcagg tatcttcgag ccagccacga tcgacattga tctggctatc ttgctgacaa
     6721 aagcaagaga acatagcgtt gccttggtag gtccagcggc ggaggaactc tttgatccgg
     6781 ttcctgaaca ggatctattt gaggcgctaa atgaaacctt aacgctatgg aactcgccgc
     6841 ccgactgggc tggcgatgag cgaaatgtag tgcttacgtt gtcccgcatt tggtacagcg
     6901 cagtaaccgg caaaatcgcg ccgaaggatg tcgctgccga ctgggcaatg gagcgcctgc
     6961 cggcccagta tcagcccgtc atacttgaag ctagacaggc ttatcttgga caagaagaag
     7021 atcgcttggc ctcgcgcgca gatcagttgg aagaatttgt ccactacgtg aaaggcgaga
     7081 tcaccaaggt agtcggcaaa taaccctcga gccacccatg accaaaatcc cttaacgtga
     7141 gttacgcgtc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     7201 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     7261 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     7321 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     7381 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     7441 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     7501 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     7561 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     7621 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     7681 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     7741 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     7801 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     7861 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     7921 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     7981 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     8041 actggaaagc gggcagtgag cgcaacgcaa ttaatacgcg taccgcgagc caggaagagt
     8101 ttgtagaaac gcaaaaaggc catccgtcag gatggccttc tgcttagttt gatgcctggc
     8161 agtttatggc gggcgtcctg cccgccaccc tccgggccgt tgcttcacaa cgttcaaatc
     8221 cgctcccggc ggatttgtcc tactcaggag agcgttcacc gacaaacaac agataaaacg
     8281 aaaggcccag tcttccgact gagcctttcg ttttatttga tgcctggcag ttccctactc
     8341 tcgcgttaac gcttgcatgg atgttttccc agtcacgacg ttgtaaaacg acggccagtc
     8401 ttaagctcgg gccc
//
