LOCUS       sequence-325638-        8841 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Gateway entry clone (attL1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8841)
  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 8841)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: pYPQ266E
FEATURES             Location/Qualifiers
     source          1..8841
                     /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"
     CDS             4604..4669
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5303..5323
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5330..5581
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHT
                     AYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5582..5635
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     terminator      5831..6083
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    6128..6251
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(6268..6287)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6269..6287)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6292..6308)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6292..6308)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             6739..7530
                     /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      7623..8211
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8112..8131
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8365..8382
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(8541..8568)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(8660..8746)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     8795..8817
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8809..8826
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8810..8826
                     /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 tcgctattgg 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 gaagaaggga gacgacggag gtggcggtac aggagggggt gggtccgctg agtatgtcag
     4441 ggcgttgttc gacttcaatg gaaacgacga ggaagatctg ccttttaaaa agggagatat
     4501 tctcaggatc agagataagc cggaagaaca atggtggaac gctgaagact ctgaaggtaa
     4561 gagaggtatg attcttgtcc cctacgtcga gaagtattcg ggtgactata aagaccacga
     4621 tggagattat aaggaccacg atatagatta taaggatgat gatgataaga gcggaatgac
     4681 cgatgcagag tacgtcagga ttcatgagaa acttgacatc tacacgttta agaaacagtt
     4741 tttcaacaac aaaaaatctg ttagtcaccg ctgttacgtg ctgttcgaat tgaaacgcag
     4801 aggtgagagg agagcctgct tttggggcta tgccgtcaac aagccgcaaa gcggcacaga
     4861 aaggggcatt cacgcggaga tatttagcat tagaaaggtc gaggaatacc ttcgggataa
     4921 tcccgggcaa ttcactatca attggtactc ttcatggtcc ccgtgtgcag attgcgctga
     4981 aaagatactg gagtggtata atcaagaact cagaggaaac ggtcacaccc tcaagatttg
     5041 ggcttgcaag ctttactacg agaaaaatgc aaggaaccag atcggcctct ggaacttgcg
     5101 cgacaacggc gtggggttga atgtgatggt ctcggagcat taccagtgct gccggaagat
     5161 attcattcag tcgtcacata atcaattgaa cgagaatagg tggctcgaaa aaaccctgaa
     5221 gcgggccgag aagtggagga gtgaactctc gataatgatc caggttaaaa tactgcatac
     5281 taccaaatct ccggcggtgg gaccgaagaa gaagcgcaag gtggggacca tgactaatct
     5341 ctcagatata atcgagaagg aaacaggaaa gcaactggtc atccaagaat cgattttgat
     5401 gcttcccgaa gaagtcgaag aagttatagg aaataagccc gagtctgaca tactggttca
     5461 cacagcgtac gatgaaagta cggacgagaa tgtcatgttg ctgacatcgg acgcacctga
     5521 atacaagcct tgggctctgg tcatacaaga tagtaacgga gaaaataaga ttaaaatgct
     5581 tgaaggaagg ggctccctgc tgacatgtgg agatgtggaa gagaatccag gaccatccag
     5641 gatgatagag caagatggtt gtacccaagt gctgcggccc ctcggatggc gggggtacag
     5701 cgccatgact ggccataata gacagtccgc tgctctcatg cctccatgca gcggatgtca
     5761 gaggagggga gctaggttct tcctgtctag acctacctgc cccgtgccct agtggcggcc
     5821 gccgacgtcc gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc
     5881 cggtcttgcg atgattatca tataatttct gttgaattac gttaagcatg taataattaa
     5941 catgtaatgc atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata
     6001 catttaatac gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc
     6061 ggtgtcatct atgttactag atcgggaatt gatcccccct cgacagcttc cggaaagggc
     6121 gaattcgcaa ctttgtatac aaaagttgaa cgagaaacgt aaaatgatat aaatatcaat
     6181 atattaaatt agattttgca taaaaaacag actacataat actgtaaaac acaacatatc
     6241 cagtcactat gccatccagc tgatatcccc tatagtgagt cgtattacat ggtcatagct
     6301 gtttcctggc agctctggcc cgtgtctcaa aatctctgat gttacattgc acaagataaa
     6361 aatatatcat catgcctcct ctggaccagc caggacagaa atgcctcgac ttcgctgcta
     6421 cccaaggttg ccgggtgacg cacaccgtgg aaacggatga aggcacgaac ccagtggaca
     6481 taagcctgtt cggttcgtaa gctgtaatgc aagtagcgta tgcgctcacg caactggtcc
     6541 agaaccttga ccgaacgcag cggtggtaac ggcgcagtgg cggttttcat ggcttgttat
     6601 gactgttttt ttggggtaca gtctatgcct cgggcatcca agcagcaagc gcgttacgcc
     6661 gtgggtcgat gtttgatgtt atggagcagc aacgatgtta cgcagcaggg cagtcgccct
     6721 aaaacaaagt taaacattat gagggaagcg gtgatcgccg aagtatcgac tcaactatca
     6781 gaggtagttg gcgtcatcga gcgccatctc gaaccgacgt tgctggccgt acatttgtac
     6841 ggctccgcag tggatggcgg cctgaagcca cacagtgata ttgatttgct ggttacggtg
     6901 accgtaaggc ttgatgaaac aacgcggcga gctttgatca acgacctttt ggaaacttcg
     6961 gcttcccctg gagagagcga gattctccgc gctgtagaag tcaccattgt tgtgcacgac
     7021 gacatcattc cgtggcgtta tccagctaag cgcgaactgc aatttggaga atggcagcgc
     7081 aatgacattc ttgcaggtat cttcgagcca gccacgatcg acattgatct ggctatcttg
     7141 ctgacaaaag caagagaaca tagcgttgcc ttggtaggtc cagcggcgga ggaactcttt
     7201 gatccggttc ctgaacagga tctatttgag gcgctaaatg aaaccttaac gctatggaac
     7261 tcgccgcccg actgggctgg cgatgagcga aatgtagtgc ttacgttgtc ccgcatttgg
     7321 tacagcgcag taaccggcaa aatcgcgccg aaggatgtcg ctgccgactg ggcaatggag
     7381 cgcctgccgg cccagtatca gcccgtcata cttgaagcta gacaggctta tcttggacaa
     7441 gaagaagatc gcttggcctc gcgcgcagat cagttggaag aatttgtcca ctacgtgaaa
     7501 ggcgagatca ccaaggtagt cggcaaataa ccctcgagcc acccatgacc aaaatccctt
     7561 aacgtgagtt acgcgtcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     7621 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     7681 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     7741 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     7801 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     7861 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     7921 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     7981 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     8041 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     8101 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     8161 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     8221 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     8281 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     8341 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     8401 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     8461 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atacgcgtac cgcgagccag
     8521 gaagagtttg tagaaacgca aaaaggccat ccgtcaggat ggccttctgc ttagtttgat
     8581 gcctggcagt ttatggcggg cgtcctgccc gccaccctcc gggccgttgc ttcacaacgt
     8641 tcaaatccgc tcccggcgga tttgtcctac tcaggagagc gttcaccgac aaacaacaga
     8701 taaaacgaaa ggcccagtct tccgactgag cctttcgttt tatttgatgc ctggcagttc
     8761 cctactctcg cgttaacgct tgcatggatg ttttcccagt cacgacgttg taaaacgacg
     8821 gccagtctta agctcgggcc c
//
