LOCUS       sequence-306141-        8984 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry clone for CRISPR-iSpyMacCas9 ABE7.10 A-G base editing 
            at NAAR PAMs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8984)
  AUTHORS   Sretenovic S, Yin D, Levav A, Selengut JD, Mount SM, Qi Y
  TITLE     Expanding plant genome-editing scope by an engineered iSpyMacCas9 
            system that targets A-rich PAM sequences.
  JOURNAL   Plant Commun. 2020 Jul 22;2(2):100101. doi: 
            10.1016/j.xplc.2020.100101. eCollection 2021 Mar 8.
  PUBMED    33898973
REFERENCE   2  (bases 1 to 8984)
  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: "Plant 
            Commun."; date: "2020-07-22"; volume: "2(2):100101. doi"; pages: " 
            10.1016/j.xplc.2020.100101. eCollection 2021 Mar 8"
COMMENT     Sequence Label: pYPQ262m3
FEATURES             Location/Qualifiers
     source          1..8984
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(143..170)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(262..348)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     397..419
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     411..428
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     412..428
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    444..543
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             583..603
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1798..1863
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1870..1890
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5992..6039
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     protein_bind    6714..6837
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(6854..6873)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6855..6873)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6878..6894)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6878..6894)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             7325..8116
                     /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      8209..8797
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8698..8717
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8951..8968
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
       61 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatacgcgt accgcgagcc
      121 aggaagagtt tgtagaaacg caaaaaggcc atccgtcagg atggccttct gcttagtttg
      181 atgcctggca gtttatggcg ggcgtcctgc ccgccaccct ccgggccgtt gcttcacaac
      241 gttcaaatcc gctcccggcg gatttgtcct actcaggaga gcgttcaccg acaaacaaca
      301 gataaaacga aaggcccagt cttccgactg agcctttcgt tttatttgat gcctggcagt
      361 tccctactct cgcgttaacg cttgcatgga tgttttccca gtcacgacgt tgtaaaacga
      421 cggccagtct taagctcggg ccccaaataa tgattttatt ttgactgata gtgacctgtt
      481 cgttgcaaca aattgatgag caatgctttt ttataatgcc aactttgtac aaaaaagcag
      541 gctccgatga aacggacagc cgacggaagc gagttcgagt caccaaagaa gaagcggaaa
      601 gtctctgaag tcgagtttag ccacgagtat tggatgaggc acgcactgac cctggcaaag
      661 cgagcatggg atgaaagaga agtccccgtg ggcgccgtgc tggtgcacaa caatagagtg
      721 atcggagagg gatggaacag gccaatcggc cgccacgacc ctaccgcaca cgcagagatc
      781 atggcactga ggcagggagg cctggtcatg cagaattacc gcctgatcga tgccaccctg
      841 tatgtgacac tggagccatg cgtgatgtgc gcaggagcaa tgatccacag caggatcgga
      901 agagtggtgt tcggagcacg ggacgccaag accggcgcag caggctccct gatggatgtg
      961 ctgcaccacc ccggcatgaa ccaccgggtg gagatcacag agggaatcct ggcagacgag
     1021 tgcgccgccc tgctgagcga tttctttaga atgcggagac aggagatcaa ggcccagaag
     1081 aaggcacaga gctccaccga ctctggagga tctagcggag gatcctctgg aagcgagaca
     1141 ccaggcacaa gcgagtccgc cacaccagag agctccggcg gctcctccgg aggatcctct
     1201 gaggtggagt tttcccacga gtactggatg agacatgccc tgaccctggc caagagggca
     1261 cgcgatgaga gggaggtgcc tgtgggagcc gtgctggtgc tgaacaatag agtgatcggc
     1321 gagggctgga acagagccat cggcctgcac gacccaacag cccatgccga aattatggcc
     1381 ctgagacagg gcggcctggt catgcagaac tacagactga ttgacgccac cctgtacgtg
     1441 acattcgagc cttgcgtgat gtgcgccggc gccatgatcc actctaggat cggccgcgtg
     1501 gtgtttggcg tgaggaacgc aaaaaccggc gccgcaggct ccctgatgga cgtgctgcac
     1561 taccccggca tgaatcaccg cgtcgaaatt accgagggaa tcctggcaga tgaatgtgcc
     1621 gccctgctgt gctatttctt tcggatgcct agacaggtgt tcaatgctca gaagaaggcc
     1681 cagagctcca ccgactccgg aggatctagc ggaggctcct ctggctctga gacacctggc
     1741 acaagcgaga gcgcaacacc tgaaagcagc gggggcagca gcggggggtc aaccatggat
     1801 tacaaggacc acgacgggga ttacaaggac cacgacattg attacaagga tgatgatgac
     1861 aagatggctc cgaagaagaa gaggaaggtt ggcatccacg gggtgccagc tgctgacaag
     1921 aagtactcga tcggcctcgc tattgggact aactctgttg gctgggccgt gatcaccgac
     1981 gagtacaagg tgccctcaaa gaagttcaag gtcctgggca acaccgatcg gcattccatc
     2041 aagaagaatc tcattggcgc tctcctgttc gacagcggcg agacggctga ggctacgcgg
     2101 ctcaagcgca ccgcccgcag gcggtacacg cgcaggaaga atcgcatctg ctacctgcag
     2161 gagattttct ccaacgagat ggcgaaggtt gacgattctt tcttccacag gctggaggag
     2221 tcattcctcg tggaggagga taagaagcac gagcggcatc caatcttcgg caacattgtc
     2281 gacgaggttg cctaccacga gaagtaccct acgatctacc atctgcggaa gaagctcgtg
     2341 gactccacag ataaggcgga cctccgcctg atctacctcg ctctggccca catgattaag
     2401 ttcaggggcc atttcctgat cgagggggat ctcaacccgg acaatagcga tgttgacaag
     2461 ctgttcatcc agctcgtgca gacgtacaac cagctcttcg aggagaaccc cattaatgcg
     2521 tcaggcgtcg acgcgaaggc tatcctgtcc gctaggctct cgaagtctcg gaagctcgag
     2581 aacctgatcg cccagctgcc gggcgagaag aagaacggcc tgttcgggaa tctcattgcg
     2641 ctcagcctgg ggctcacgcc caacttcaag tcgaatttcg atctcgctga ggacgccaag
     2701 ctgcagctct ccaaggacac atacgacgat gacctggata acctcctggc ccagatcggc
     2761 gatcagtacg cggacctgtt cctcgctgcc aagaatctgt cggacgccat cctcctgtct
     2821 gatattctca gggtgaacac cgagattacg aaggctccgc tctcagcctc catgatcaag
     2881 cgctacgacg agcaccatca ggatctgacc ctcctgaagg cgctggtcag gcagcagctc
     2941 cccgagaagt acaaggagat cttcttcgat cagtcgaaga acggctacgc tgggtacatt
     3001 gacggcgggg cctctcagga ggagttctac aagttcatca agccgattct ggagaagatg
     3061 gacggcacgg aggagctgct ggtgaagctc aagcgcgagg acctcctgag gaagcagcgg
     3121 acattcgata acggcagcat cccacaccag attcatctcg gggagctgca cgctatcctg
     3181 aggaggcagg aggacttcta ccctttcctc aaggataacc gcgagaagat cgagaagatt
     3241 ctgactttca ggatcccgta ctacgtcggc ccactcgcta ggggcaactc ccgcttcgct
     3301 tggatgaccc gcaagtcaga ggagacgatc acgccgtgga acttcgagga ggtggtcgac
     3361 aagggcgcta gcgctcagtc gttcatcgag aggatgacga atttcgacaa gaacctgcca
     3421 aatgagaagg tgctccctaa gcactcgctc ctgtacgagt acttcacagt ctacaacgag
     3481 ctgactaagg tgaagtatgt gaccgagggc atgaggaagc cggctttcct gtctggggag
     3541 cagaagaagg ccatcgtgga cctcctgttc aagaccaacc ggaaggtcac ggttaagcag
     3601 ctcaaggagg actacttcaa gaagattgag tgcttcgatt cggtcgagat ctctggcgtt
     3661 gaggaccgct tcaacgcctc cctggggacc taccacgatc tcctgaagat cattaaggat
     3721 aaggacttcc tggacaacga ggagaatgag gatatcctcg aggacattgt gctgacactc
     3781 actctgttcg aggaccggga gatgatcgag gagcgcctga agacttacgc ccatctcttc
     3841 gatgacaagg tcatgaagca gctcaagagg aggaggtaca ccggctgggg gaggctgagc
     3901 aggaagctca tcaacggcat tcgggacaag cagtccggga agacgatcct cgacttcctg
     3961 aagagcgatg gcttcgcgaa ccgcaatttc atgcagctga ttcacgatga cagcctcaca
     4021 ttcaaggagg atatccagaa ggctcaggtg agcggccagg gggactcgct gcacgagcat
     4081 atcgcgaacc tcgctggctc gccagctatc aagaagggga ttctgcagac cgtgaaggtt
     4141 gtggacgagc tggtgaaggt catgggcagg cacaagcctg agaacatcgt cattgagatg
     4201 gcccgggaga atcagaccac gcagaagggc cagaagaact cacgcgagag gatgaagagg
     4261 atcgaggagg gcattaagga gctggggtcc cagatcctca aggagcaccc ggtggagaac
     4321 acgcagctgc agaatgagaa gctctacctg tactacctcc agaatggccg cgatatgtat
     4381 gtggaccagg agctggatat taacaggctc agcgattacg acgtcgatca tatcgttcca
     4441 cagtcattcc tgaaggatga ctccattgac aacaaggtcc tcaccaggtc ggacaagaac
     4501 cggggcaagt ctgataatgt tccttcagag gaggtcgtta agaagatgaa gaactactgg
     4561 cgccagctcc tgaatgccaa gctgatcacg cagcggaagt tcgataacct cacaaaggct
     4621 gagaggggcg ggctctctga gctggacaag gcgggcttca tcaagaggca gctggtcgag
     4681 acacggcaga tcactaagca cgttgcgcag attctcgact cacggatgaa cactaagtac
     4741 gatgagaatg acaagctgat ccgcgaggtg aaggtcatca ccctgaagtc aaagctcgtc
     4801 tccgacttca ggaaggattt ccagttctac aaggttcggg agatcaacaa ttaccaccat
     4861 gcccatgacg cgtacctgaa cgcggtggtc ggcacagctc tgatcaagaa gtacccaaag
     4921 ctcgagagcg agttcgtgta cggggactac aaggtttacg atgtgaggaa gatgatcgcc
     4981 aagtcggagc aggagattgg caaggctacc gccaagtact tcttctactc taacattatg
     5041 aatttcttca agacagagat cactctggcc aatggcgaga tccggaagcg ccccctcatc
     5101 gagacgaacg gcgagacggg ggagatcgtg tgggacaagg gcagggattt cgcgaccgtc
     5161 aggaaggttc tctccatgcc acaagtgaat atcgtcaaga agacagagat acaaacggtt
     5221 ggtcagaacg ggggtctttt cgatgataac ccaaaatccc cgcttgaggt gaccccttca
     5281 aagctggtgc cactcaaaaa ggaattgaat ccaaaaaagt atggcggata ccaaaagcct
     5341 acaacggcgt atcccgtcct cttgattacg gatacaaagc aacttattcc tatatccgtt
     5401 atgaataaaa aacagttcga gcagaaccct gttaagttcc tgcgggatag aggctatcaa
     5461 caagtgggca agaacgattt tattaagctt cctaaatata cgctcgtgga tattggagac
     5521 ggcataaaaa gactttgggc ttcctcgaaa gaaatacata agggcaacca actcgtcgtc
     5581 tcaaaaaagt ctcagatcct tctttatcat gcgcaccacc ttgattcaga cctctcaaac
     5641 gattatcttc agaaccataa tcagcagttt gacgtgctct tcaacgagat aatttcattc
     5701 tctaaaaagt gcaagctcgg gaaggagcac atacagaaaa tcgaaaacgt ctattctaac
     5761 aaaaagaact cagcctccat agaggagttg gcagaatcat tcataaagct cctgggcttt
     5821 actcagctgg gcgccacatc accgtttaac tttctgggag tcaaactcaa tcaaaagcag
     5881 tataagggca aaaaggacta tattctcccc tgtaccgaag gaactctcat caggcaatcc
     5941 attacagggt tgtacgaaac aagggtcgac ctctcaaaaa taggagagga caagcggcca
     6001 gcggcgacga agaaggcggg gcaggcgaag aagaagaagt gagctcagag ctttcgttcg
     6061 tatcatcggt ttcgacaacg ttcgtcaagt tcaatgcatc agtttcattg cgcacacacc
     6121 agaatcctac tgagtttgag tattatggca ttgggaaaac tgtttttctt gtaccatttg
     6181 ttgtgcttgt aatttactgt gttttttatt cggttttcgc tatcgaactg tgaaatggaa
     6241 atggatggag aagagttaat gaatgatatg gtccttttgt tcattctcaa attaatatta
     6301 tttgtttttt ctcttatttg ttgtgtgttg aatttgaaat tataagagat atgcaaacat
     6361 tttgttttga gtaaaaatgt gtcaaatcgt ggcctctaat gaccgaagtt aatatgagga
     6421 gtaaaacact tgtagttgta ccattatgct tattcactag gcaacaaata tattttcaga
     6481 cctagaaaag ctgcaaatgt tactgaatac aagtatgtcc tcttgtgttt tagacattta
     6541 tgaactttcc tttatgtaat tttccagaat ccttgtcaga ttctaatcat tgctttataa
     6601 ttatagttat actcatggat ttgtagttga gtatgaaaat attttttaat gcattttatg
     6661 acttgccaat tgttccggaa ctctagataa gcttaccgga aagggcgaat tcgcaacttt
     6721 gtatacaaaa gttgaacgag aaacgtaaaa tgatataaat atcaatatat taaattagat
     6781 tttgcataaa aaacagacta cataatactg taaaacacaa catatccagt cactatgcca
     6841 tccagctgat atcccctata gtgagtcgta ttacatggtc atagctgttt cctggcagct
     6901 ctggcccgtg tctcaaaatc tctgatgtta cattgcacaa gataaaaata tatcatcatg
     6961 cctcctctgg accagccagg acagaaatgc ctcgacttcg ctgctaccca aggttgccgg
     7021 gtgacgcaca ccgtggaaac ggatgaaggc acgaacccag tggacataag cctgttcggt
     7081 tcgtaagctg taatgcaagt agcgtatgcg ctcacgcaac tggtccagaa ccttgaccga
     7141 acgcagcggt ggtaacggcg cagtggcggt tttcatggct tgttatgact gtttttttgg
     7201 ggtacagtct atgcctcggg catccaagca gcaagcgcgt tacgccgtgg gtcgatgttt
     7261 gatgttatgg agcagcaacg atgttacgca gcagggcagt cgccctaaaa caaagttaaa
     7321 cattatgagg gaagcggtga tcgccgaagt atcgactcaa ctatcagagg tagttggcgt
     7381 catcgagcgc catctcgaac cgacgttgct ggccgtacat ttgtacggct ccgcagtgga
     7441 tggcggcctg aagccacaca gtgatattga tttgctggtt acggtgaccg taaggcttga
     7501 tgaaacaacg cggcgagctt tgatcaacga ccttttggaa acttcggctt cccctggaga
     7561 gagcgagatt ctccgcgctg tagaagtcac cattgttgtg cacgacgaca tcattccgtg
     7621 gcgttatcca gctaagcgcg aactgcaatt tggagaatgg cagcgcaatg acattcttgc
     7681 aggtatcttc gagccagcca cgatcgacat tgatctggct atcttgctga caaaagcaag
     7741 agaacatagc gttgccttgg taggtccagc ggcggaggaa ctctttgatc cggttcctga
     7801 acaggatcta tttgaggcgc taaatgaaac cttaacgcta tggaactcgc cgcccgactg
     7861 ggctggcgat gagcgaaatg tagtgcttac gttgtcccgc atttggtaca gcgcagtaac
     7921 cggcaaaatc gcgccgaagg atgtcgctgc cgactgggca atggagcgcc tgccggccca
     7981 gtatcagccc gtcatacttg aagctagaca ggcttatctt ggacaagaag aagatcgctt
     8041 ggcctcgcgc gcagatcagt tggaagaatt tgtccactac gtgaaaggcg agatcaccaa
     8101 ggtagtcggc aaataaccct cgagccaccc atgaccaaaa tcccttaacg tgagttacgc
     8161 gtcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     8221 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     8281 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     8341 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     8401 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     8461 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     8521 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     8581 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     8641 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     8701 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     8761 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     8821 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     8881 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     8941 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcc
//
