LOCUS       sequence-325640-        7775 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry plasmid (attL1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7775)
  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 7775)
  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: pYPQ166-SpRY
FEATURES             Location/Qualifiers
     source          1..7775
                     /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             122..187
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             194..214
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     RBS             4252..4260
                     /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             4340..4387
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     protein_bind    5062..5185
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(5202..5221)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5203..5221)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5226..5242)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5226..5242)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5673..6464
                     /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      6557..7145
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7046..7065
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7299..7316
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(7475..7502)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(7594..7680)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     7729..7751
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7743..7760
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7744..7760
                     /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 ccgaattcgc ccttcaccat
      121 ggattacaag gaccacgacg gggattacaa ggaccacgac attgattaca aggatgatga
      181 tgacaagatg gctccgaaga agaagaggaa ggttggcatc cacggggtgc cagctgctga
      241 caagaagtac tcgatcggcc tcgatattgg gactaactct gttggctggg ccgtgatcac
      301 cgacgagtac aaggtgccct caaagaagtt caaggtcctg ggcaacaccg atcggcattc
      361 catcaagaag aatctcattg gcgctctcct gttcgacagc ggcgagacgg ctgagaggac
      421 gcggctcaag cgcaccgccc gcaggcggta cacgcgcagg aagaatcgca tctgctacct
      481 gcaggagatt ttctccaacg agatggcgaa ggttgacgat tctttcttcc acaggctgga
      541 ggagtcattc ctcgtggagg aggataagaa gcacgagcgg catccaatct tcggcaacat
      601 tgtcgacgag gttgcctacc acgagaagta ccctacgatc taccatctgc ggaagaagct
      661 cgtggactcc acagataagg cggacctccg cctgatctac ctcgctctgg cccacatgat
      721 taagttcagg ggccatttcc tgatcgaggg ggatctcaac ccggacaata gcgatgttga
      781 caagctgttc atccagctcg tgcagacgta caaccagctc ttcgaggaga accccattaa
      841 tgcgtcaggc gtcgacgcga aggctatcct gtccgctagg ctctcgaagt ctcggcgcct
      901 cgagaacctg atcgcccagc tgccgggcga gaagaagaac ggcctgttcg ggaatctcat
      961 tgcgctcagc ctggggctca cgcccaactt caagtcgaat ttcgatctcg ctgaggacgc
     1021 caagctgcag ctctccaagg acacatacga cgatgacctg gataacctcc tggcccagat
     1081 cggcgatcag tacgcggacc tgttcctcgc tgccaagaat ctgtcggacg ccatcctcct
     1141 gtctgatatt ctcagggtga acaccgagat tacgaaggct ccgctctcag cctccatgat
     1201 caagcgctac gacgagcacc atcaggatct gaccctcctg aaggcgctgg tcaggcagca
     1261 gctccccgag aagtacaagg agatcttctt cgatcagtcg aagaacggct acgctgggta
     1321 cattgacggc ggggcctctc aggaggagtt ctacaagttc atcaagccga ttctggagaa
     1381 gatggacggc acggaggagc tgctggtgaa gctcaatcgc gaggacctcc tgaggaagca
     1441 gcggacattc gataacggca gcatcccaca ccagattcat ctcggggagc tgcacgctat
     1501 cctgaggagg caggaggact tctacccttt cctcaaggat aaccgcgaga agatcgagaa
     1561 gattctgact ttcaggatcc cgtactacgt cggcccactc gctaggggca actcccgctt
     1621 cgcttggatg acccgcaagt cagaggagac gatcacgccg tggaacttcg aggaggtggt
     1681 cgacaagggc gctagcgctc agtcgttcat cgagaggatg acgaatttcg acaagaacct
     1741 gccaaatgag aaggtgctcc ctaagcactc gctcctgtac gagtacttca cagtctacaa
     1801 cgagctgact aaggtgaagt atgtgaccga gggcatgagg aagccggctt tcctgtctgg
     1861 ggagcagaag aaggccatcg tggacctcct gttcaagacc aaccggaagg tcacggttaa
     1921 gcagctcaag gaggactact tcaagaagat tgagtgcttc gattcggtcg agatctctgg
     1981 cgttgaggac cgcttcaacg cctccctggg gacctaccac gatctcctga agatcattaa
     2041 ggataaggac ttcctggaca acgaggagaa tgaggatatc ctcgaggaca ttgtgctgac
     2101 actcactctg ttcgaggacc gggagatgat cgaggagcgc ctgaagactt acgcccatct
     2161 cttcgatgac aaggtcatga agcagctcaa gaggaggagg tacaccggct gggggaggct
     2221 gagcaggaag ctcatcaacg gcattcggga caagcagtcc gggaagacga tcctcgactt
     2281 cctgaagagc gatggcttcg cgaaccgcaa tttcatgcag ctgattcacg atgacagcct
     2341 cacattcaag gaggatatcc agaaggctca ggtgagcggc cagggggact cgctgcacga
     2401 gcatatcgcg aacctcgctg gctcgccagc tatcaagaag gggattctgc agaccgtgaa
     2461 ggttgtggac gagctggtga aggtcatggg caggcacaag cctgagaaca tcgtcattga
     2521 gatggcccgg gagaatcaga ccacgcagaa gggccagaag aactcacgcg agaggatgaa
     2581 gaggatcgag gagggcatta aggagctggg gtcccagatc ctcaaggagc acccggtgga
     2641 gaacacgcag ctgcagaatg agaagctcta cctgtactac ctccagaatg gccgcgatat
     2701 gtatgtggac caggagctgg atattaacag gctcagcgat tacgacgtcg atcatatcgt
     2761 tccacagtca ttcctgaagg atgactccat tgacaacaag gtcctcacca ggtcggacaa
     2821 gaaccggggc aagtctgata atgttccttc agaggaggtc gttaagaaga tgaagaacta
     2881 ctggcgccag ctcctgaatg ccaagctgat cacgcagcgg aagttcgata acctcacaaa
     2941 ggctgagagg ggcgggctct ctgagctgga caaggcgggc ttcatcaaga ggcagctggt
     3001 cgagacacgg cagatcacta agcacgttgc gcagattctc gactcacgga tgaacactaa
     3061 gtacgatgag aatgacaagc tgatccgcga ggtgaaggtc atcaccctga agtcaaagct
     3121 cgtctccgac ttcaggaagg atttccagtt ctacaaggtt cgggagatca acaattacca
     3181 ccatgcccat gacgcgtacc tgaacgcggt ggtcggcaca gctctgatca agaagtaccc
     3241 aaagctcgag agcgagttcg tgtacgggga ctacaaggtt tacgatgtga ggaagatgat
     3301 cgccaagtcg gagcaggaga ttggcaaggc taccgccaag tacttcttct actctaacat
     3361 tatgaatttc ttcaagacag agatcactct ggccaatggc gagatccgga agcgccccct
     3421 catcgagacg aacggcgaga cgggggagat cgtgtgggac aagggcaggg atttcgcgac
     3481 cgtcaggaag gttctctcca tgccacaagt gaatatcgtc aagaagacag aggtccagac
     3541 tggcgggttc tctaaggagt caattaggcc taagcggaac agcgacaagc tcatcgcccg
     3601 caagaaggac tgggatccga agaagtacgg cgggttcctg tggcccactg tggcctactc
     3661 ggtcctggtt gtggcgaagg ttgagaaggg caagtccaag aagctcaaga gcgtgaagga
     3721 gctgctgggg atcacgatta tggagcgctc cagcttcgag aagaacccga tcgatttcct
     3781 ggaggcgaag ggctacaagg aggtgaagaa ggacctgatc attaagctcc ccaagtactc
     3841 actcttcgag ctggagaacg gcaggaagcg gatgctggct tccgctaagc agctgcagaa
     3901 ggggaacgag ctggctctgc cgtccaagta tgtgaacttc ctctacctgg cctcccacta
     3961 cgagaagctc aagggcagcc ccgaggacaa cgagcagaag cagctgttcg tcgagcagca
     4021 caagcattac ctcgacgaga tcattgagca gatttccgag ttctccaagc gcgtgatcct
     4081 ggccgacgcg aatctggata aggtcctctc cgcgtacaac aagcaccgcg acaagccaat
     4141 cagggagcag gctgagaata tcattcatct cttcaccctg acgaggctcg gcgcccctag
     4201 ggctttcaag tacttcgaca caactatcga tccgaagcag tacaggagca ctaaggaggt
     4261 cctggacgcg accctcatcc accagtcgat taccggcctc tacgagacgc gcatcgacct
     4321 gtctcagctc gggggcgaca agcggccagc ggcgacgaag aaggcggggc aggcgaagaa
     4381 gaagaagtga gctcagagct ttcgttcgta tcatcggttt cgacaacgtt cgtcaagttc
     4441 aatgcatcag tttcattgcg cacacaccag aatcctactg agtttgagta ttatggcatt
     4501 gggaaaactg tttttcttgt accatttgtt gtgcttgtaa tttactgtgt tttttattcg
     4561 gttttcgcta tcgaactgtg aaatggaaat ggatggagaa gagttaatga atgatatggt
     4621 ccttttgttc attctcaaat taatattatt tgttttttct cttatttgtt gtgtgttgaa
     4681 tttgaaatta taagagatat gcaaacattt tgttttgagt aaaaatgtgt caaatcgtgg
     4741 cctctaatga ccgaagttaa tatgaggagt aaaacacttg tagttgtacc attatgctta
     4801 ttcactaggc aacaaatata ttttcagacc tagaaaagct gcaaatgtta ctgaatacaa
     4861 gtatgtcctc ttgtgtttta gacatttatg aactttcctt tatgtaattt tccagaatcc
     4921 ttgtcagatt ctaatcattg ctttataatt atagttatac tcatggattt gtagttgagt
     4981 atgaaaatat tttttaatgc attttatgac ttgccaattg ttccggaact ctagataagc
     5041 ttaccggaaa gggcgaattc gcaactttgt atacaaaagt tgaacgagaa acgtaaaatg
     5101 atataaatat caatatatta aattagattt tgcataaaaa acagactaca taatactgta
     5161 aaacacaaca tatccagtca ctatgccatc cagctgatat cccctatagt gagtcgtatt
     5221 acatggtcat agctgtttcc tggcagctct ggcccgtgtc tcaaaatctc tgatgttaca
     5281 ttgcacaaga taaaaatata tcatcatgcc tcctctggac cagccaggac agaaatgcct
     5341 cgacttcgct gctacccaag gttgccgggt gacgcacacc gtggaaacgg atgaaggcac
     5401 gaacccagtg gacataagcc tgttcggttc gtaagctgta atgcaagtag cgtatgcgct
     5461 cacgcaactg gtccagaacc ttgaccgaac gcagcggtgg taacggcgca gtggcggttt
     5521 tcatggcttg ttatgactgt ttttttgggg tacagtctat gcctcgggca tccaagcagc
     5581 aagcgcgtta cgccgtgggt cgatgtttga tgttatggag cagcaacgat gttacgcagc
     5641 agggcagtcg ccctaaaaca aagttaaaca ttatgaggga agcggtgatc gccgaagtat
     5701 cgactcaact atcagaggta gttggcgtca tcgagcgcca tctcgaaccg acgttgctgg
     5761 ccgtacattt gtacggctcc gcagtggatg gcggcctgaa gccacacagt gatattgatt
     5821 tgctggttac ggtgaccgta aggcttgatg aaacaacgcg gcgagctttg atcaacgacc
     5881 ttttggaaac ttcggcttcc cctggagaga gcgagattct ccgcgctgta gaagtcacca
     5941 ttgttgtgca cgacgacatc attccgtggc gttatccagc taagcgcgaa ctgcaatttg
     6001 gagaatggca gcgcaatgac attcttgcag gtatcttcga gccagccacg atcgacattg
     6061 atctggctat cttgctgaca aaagcaagag aacatagcgt tgccttggta ggtccagcgg
     6121 cggaggaact ctttgatccg gttcctgaac aggatctatt tgaggcgcta aatgaaacct
     6181 taacgctatg gaactcgccg cccgactggg ctggcgatga gcgaaatgta gtgcttacgt
     6241 tgtcccgcat ttggtacagc gcagtaaccg gcaaaatcgc gccgaaggat gtcgctgccg
     6301 actgggcaat ggagcgcctg ccggcccagt atcagcccgt catacttgaa gctagacagg
     6361 cttatcttgg acaagaagaa gatcgcttgg cctcgcgcgc agatcagttg gaagaatttg
     6421 tccactacgt gaaaggcgag atcaccaagg tagtcggcaa ataaccctcg agccacccat
     6481 gaccaaaatc ccttaacgtg agttacgcgt cgttccactg agcgtcagac cccgtagaaa
     6541 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     6601 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     6661 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     6721 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     6781 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     6841 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     6901 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     6961 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     7021 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     7081 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     7141 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     7201 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     7261 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     7321 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     7381 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatacgc
     7441 gtaccgcgag ccaggaagag tttgtagaaa cgcaaaaagg ccatccgtca ggatggcctt
     7501 ctgcttagtt tgatgcctgg cagtttatgg cgggcgtcct gcccgccacc ctccgggccg
     7561 ttgcttcaca acgttcaaat ccgctcccgg cggatttgtc ctactcagga gagcgttcac
     7621 cgacaaacaa cagataaaac gaaaggccca gtcttccgac tgagcctttc gttttatttg
     7681 atgcctggca gttccctact ctcgcgttaa cgcttgcatg gatgttttcc cagtcacgac
     7741 gttgtaaaac gacggccagt cttaagctcg ggccc
//
