LOCUS       sequence-308763-        8331 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry clone for CRISPR-iSpyMacCas9 hAID C-T base editing at 
            NAAR PAMs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8331)
  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 8331)
  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: pYPQ265H
FEATURES             Location/Qualifiers
     source          1..8331
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     16..33
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(192..219)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(311..397)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     446..468
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     460..477
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     461..477
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    493..592
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             1247..1312
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1319..1339
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5441..5488
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5510..5758
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5771..5791
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      5813..6065
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    6110..6233
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(6250..6269)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6251..6269)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6274..6290)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6274..6290)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             6721..7512
                     /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      7605..8193
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8094..8113
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
       61 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
      121 tggaaagcgg gcagtgagcg caacgcaatt aatacgcgta ccgcgagcca ggaagagttt
      181 gtagaaacgc aaaaaggcca tccgtcagga tggccttctg cttagtttga tgcctggcag
      241 tttatggcgg gcgtcctgcc cgccaccctc cgggccgttg cttcacaacg ttcaaatccg
      301 ctcccggcgg atttgtccta ctcaggagag cgttcaccga caaacaacag ataaaacgaa
      361 aggcccagtc ttccgactga gcctttcgtt ttatttgatg cctggcagtt ccctactctc
      421 gcgttaacgc ttgcatggat gttttcccag tcacgacgtt gtaaaacgac ggccagtctt
      481 aagctcgggc cccaaataat gattttattt tgactgatag tgacctgttc gttgcaacaa
      541 attgatgagc aatgcttttt tataatgcca actttgtaca aaaaagcagg ctccgaattc
      601 gcccttcatg gactcacttt tgatgaatag gcgggagttc ttgtaccagt ttaagaacgt
      661 gcggtgggcg aaaggtcgga gggaaaccta cctttgctat gtggttaaga gacgcgacag
      721 cgcaacatcc ttctccttgg actttggata ccttcgcaat aagaatgggt gtcatgtcga
      781 actgttgttt ctccggtata ttagcgattg ggacctggac cccggcaggt gttacagggt
      841 gacatggttc atatcgtggt ctccatgcta tgactgcgct aggcatgttg ctgattttct
      901 taggggcaat ccaaacctga gtcttcgcat tttcacggcg cggctgtatt tttgtgagga
      961 ccgcaaggca gagcccgagg gattgcgcag gcttcatagg gcaggagtcc aaatagctat
     1021 catgactttc aaagactatt tttactgttg gaacacgttc gtcgagaacc acggacggac
     1081 gtttaaggct tgggaaggac tgcatgaaaa cagtgtcagg ctctcaaggc aactccgccg
     1141 catacttttg cctttgtacg aggttgacga cctgcgcgac gcatttcgga cttcaggttc
     1201 tgagacccct ggcacaagtg agtcagcaac acccgagtcc accatggatt acaaggacca
     1261 cgacggggat tacaaggacc acgacattga ttacaaggat gatgatgaca agatggctcc
     1321 gaagaagaag aggaaggttg gcatccacgg ggtgccagct gctgacaaga agtactcgat
     1381 cggcctcgct attgggacta actctgttgg ctgggccgtg atcaccgacg agtacaaggt
     1441 gccctcaaag aagttcaagg tcctgggcaa caccgatcgg cattccatca agaagaatct
     1501 cattggcgct ctcctgttcg acagcggcga gacggctgag gctacgcggc tcaagcgcac
     1561 cgcccgcagg cggtacacgc gcaggaagaa tcgcatctgc tacctgcagg agattttctc
     1621 caacgagatg gcgaaggttg acgattcttt cttccacagg ctggaggagt cattcctcgt
     1681 ggaggaggat aagaagcacg agcggcatcc aatcttcggc aacattgtcg acgaggttgc
     1741 ctaccacgag aagtacccta cgatctacca tctgcggaag aagctcgtgg actccacaga
     1801 taaggcggac ctccgcctga tctacctcgc tctggcccac atgattaagt tcaggggcca
     1861 tttcctgatc gagggggatc tcaacccgga caatagcgat gttgacaagc tgttcatcca
     1921 gctcgtgcag acgtacaacc agctcttcga ggagaacccc attaatgcgt caggcgtcga
     1981 cgcgaaggct atcctgtccg ctaggctctc gaagtctcgg aagctcgaga acctgatcgc
     2041 ccagctgccg ggcgagaaga agaacggcct gttcgggaat ctcattgcgc tcagcctggg
     2101 gctcacgccc aacttcaagt cgaatttcga tctcgctgag gacgccaagc tgcagctctc
     2161 caaggacaca tacgacgatg acctggataa cctcctggcc cagatcggcg atcagtacgc
     2221 ggacctgttc ctcgctgcca agaatctgtc ggacgccatc ctcctgtctg atattctcag
     2281 ggtgaacacc gagattacga aggctccgct ctcagcctcc atgatcaagc gctacgacga
     2341 gcaccatcag gatctgaccc tcctgaaggc gctggtcagg cagcagctcc ccgagaagta
     2401 caaggagatc ttcttcgatc agtcgaagaa cggctacgct gggtacattg acggcggggc
     2461 ctctcaggag gagttctaca agttcatcaa gccgattctg gagaagatgg acggcacgga
     2521 ggagctgctg gtgaagctca agcgcgagga cctcctgagg aagcagcgga cattcgataa
     2581 cggcagcatc ccacaccaga ttcatctcgg ggagctgcac gctatcctga ggaggcagga
     2641 ggacttctac cctttcctca aggataaccg cgagaagatc gagaagattc tgactttcag
     2701 gatcccgtac tacgtcggcc cactcgctag gggcaactcc cgcttcgctt ggatgacccg
     2761 caagtcagag gagacgatca cgccgtggaa cttcgaggag gtggtcgaca agggcgctag
     2821 cgctcagtcg ttcatcgaga ggatgacgaa tttcgacaag aacctgccaa atgagaaggt
     2881 gctccctaag cactcgctcc tgtacgagta cttcacagtc tacaacgagc tgactaaggt
     2941 gaagtatgtg accgagggca tgaggaagcc ggctttcctg tctggggagc agaagaaggc
     3001 catcgtggac ctcctgttca agaccaaccg gaaggtcacg gttaagcagc tcaaggagga
     3061 ctacttcaag aagattgagt gcttcgattc ggtcgagatc tctggcgttg aggaccgctt
     3121 caacgcctcc ctggggacct accacgatct cctgaagatc attaaggata aggacttcct
     3181 ggacaacgag gagaatgagg atatcctcga ggacattgtg ctgacactca ctctgttcga
     3241 ggaccgggag atgatcgagg agcgcctgaa gacttacgcc catctcttcg atgacaaggt
     3301 catgaagcag ctcaagagga ggaggtacac cggctggggg aggctgagca ggaagctcat
     3361 caacggcatt cgggacaagc agtccgggaa gacgatcctc gacttcctga agagcgatgg
     3421 cttcgcgaac cgcaatttca tgcagctgat tcacgatgac agcctcacat tcaaggagga
     3481 tatccagaag gctcaggtga gcggccaggg ggactcgctg cacgagcata tcgcgaacct
     3541 cgctggctcg ccagctatca agaaggggat tctgcagacc gtgaaggttg tggacgagct
     3601 ggtgaaggtc atgggcaggc acaagcctga gaacatcgtc attgagatgg cccgggagaa
     3661 tcagaccacg cagaagggcc agaagaactc acgcgagagg atgaagagga tcgaggaggg
     3721 cattaaggag ctggggtccc agatcctcaa ggagcacccg gtggagaaca cgcagctgca
     3781 gaatgagaag ctctacctgt actacctcca gaatggccgc gatatgtatg tggaccagga
     3841 gctggatatt aacaggctca gcgattacga cgtcgatcat atcgttccac agtcattcct
     3901 gaaggatgac tccattgaca acaaggtcct caccaggtcg gacaagaacc ggggcaagtc
     3961 tgataatgtt ccttcagagg aggtcgttaa gaagatgaag aactactggc gccagctcct
     4021 gaatgccaag ctgatcacgc agcggaagtt cgataacctc acaaaggctg agaggggcgg
     4081 gctctctgag ctggacaagg cgggcttcat caagaggcag ctggtcgaga cacggcagat
     4141 cactaagcac gttgcgcaga ttctcgactc acggatgaac actaagtacg atgagaatga
     4201 caagctgatc cgcgaggtga aggtcatcac cctgaagtca aagctcgtct ccgacttcag
     4261 gaaggatttc cagttctaca aggttcggga gatcaacaat taccaccatg cccatgacgc
     4321 gtacctgaac gcggtggtcg gcacagctct gatcaagaag tacccaaagc tcgagagcga
     4381 gttcgtgtac ggggactaca aggtttacga tgtgaggaag atgatcgcca agtcggagca
     4441 ggagattggc aaggctaccg ccaagtactt cttctactct aacattatga atttcttcaa
     4501 gacagagatc actctggcca atggcgagat ccggaagcgc cccctcatcg agacgaacgg
     4561 cgagacgggg gagatcgtgt gggacaaggg cagggatttc gcgaccgtca ggaaggttct
     4621 ctccatgcca caagtgaata tcgtcaagaa gacagagata caaacggttg gtcagaacgg
     4681 gggtcttttc gatgataacc caaaatcccc gcttgaggtg accccttcaa agctggtgcc
     4741 actcaaaaag gaattgaatc caaaaaagta tggcggatac caaaagccta caacggcgta
     4801 tcccgtcctc ttgattacgg atacaaagca acttattcct atatccgtta tgaataaaaa
     4861 acagttcgag cagaaccctg ttaagttcct gcgggataga ggctatcaac aagtgggcaa
     4921 gaacgatttt attaagcttc ctaaatatac gctcgtggat attggagacg gcataaaaag
     4981 actttgggct tcctcgaaag aaatacataa gggcaaccaa ctcgtcgtct caaaaaagtc
     5041 tcagatcctt ctttatcatg cgcaccacct tgattcagac ctctcaaacg attatcttca
     5101 gaaccataat cagcagtttg acgtgctctt caacgagata atttcattct ctaaaaagtg
     5161 caagctcggg aaggagcaca tacagaaaat cgaaaacgtc tattctaaca aaaagaactc
     5221 agcctccata gaggagttgg cagaatcatt cataaagctc ctgggcttta ctcagctggg
     5281 cgccacatca ccgtttaact ttctgggagt caaactcaat caaaagcagt ataagggcaa
     5341 aaaggactat attctcccct gtaccgaagg aactctcatc aggcaatcca ttacagggtt
     5401 gtacgaaaca agggtcgacc tctcaaaaat aggagaggac aagcggccag cggcgacgaa
     5461 gaaggcgggg caggcgaaga agaagaaggg agacggatca ggcgggtcaa caaacctcag
     5521 tgacatcata gagaaggaaa ctggtaagca actggttatt caagagagca ttctgatgct
     5581 ccctgaggaa gtcgaggaag ttataggaaa caagcctgag agcgatatac tcgtgcatac
     5641 ggcgtatgac gagagcacgg atgaaaatgt gatgctcttg accagcgatg cgccagaata
     5701 taaaccctgg gcactggtta ttcaggactc taacggggaa aataaaatca agatgctctc
     5761 aggtggctcc ccaaagaaga aacgcaaggt ttagtggcgg ccgccgacgt ccgatcgttc
     5821 aaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg cgatgattat
     5881 catataattt ctgttgaatt acgttaagca tgtaataatt aacatgtaat gcatgacgtt
     5941 atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat acgcgataga
     6001 aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat ctatgttact
     6061 agatcgggaa ttgatccccc ctcgacagct tccggaaagg gcgaattcgc aactttgtat
     6121 acaaaagttg aacgagaaac gtaaaatgat ataaatatca atatattaaa ttagattttg
     6181 cataaaaaac agactacata atactgtaaa acacaacata tccagtcact atgccatcca
     6241 gctgatatcc cctatagtga gtcgtattac atggtcatag ctgtttcctg gcagctctgg
     6301 cccgtgtctc aaaatctctg atgttacatt gcacaagata aaaatatatc atcatgcctc
     6361 ctctggacca gccaggacag aaatgcctcg acttcgctgc tacccaaggt tgccgggtga
     6421 cgcacaccgt ggaaacggat gaaggcacga acccagtgga cataagcctg ttcggttcgt
     6481 aagctgtaat gcaagtagcg tatgcgctca cgcaactggt ccagaacctt gaccgaacgc
     6541 agcggtggta acggcgcagt ggcggttttc atggcttgtt atgactgttt ttttggggta
     6601 cagtctatgc ctcgggcatc caagcagcaa gcgcgttacg ccgtgggtcg atgtttgatg
     6661 ttatggagca gcaacgatgt tacgcagcag ggcagtcgcc ctaaaacaaa gttaaacatt
     6721 atgagggaag cggtgatcgc cgaagtatcg actcaactat cagaggtagt tggcgtcatc
     6781 gagcgccatc tcgaaccgac gttgctggcc gtacatttgt acggctccgc agtggatggc
     6841 ggcctgaagc cacacagtga tattgatttg ctggttacgg tgaccgtaag gcttgatgaa
     6901 acaacgcggc gagctttgat caacgacctt ttggaaactt cggcttcccc tggagagagc
     6961 gagattctcc gcgctgtaga agtcaccatt gttgtgcacg acgacatcat tccgtggcgt
     7021 tatccagcta agcgcgaact gcaatttgga gaatggcagc gcaatgacat tcttgcaggt
     7081 atcttcgagc cagccacgat cgacattgat ctggctatct tgctgacaaa agcaagagaa
     7141 catagcgttg ccttggtagg tccagcggcg gaggaactct ttgatccggt tcctgaacag
     7201 gatctatttg aggcgctaaa tgaaacctta acgctatgga actcgccgcc cgactgggct
     7261 ggcgatgagc gaaatgtagt gcttacgttg tcccgcattt ggtacagcgc agtaaccggc
     7321 aaaatcgcgc cgaaggatgt cgctgccgac tgggcaatgg agcgcctgcc ggcccagtat
     7381 cagcccgtca tacttgaagc tagacaggct tatcttggac aagaagaaga tcgcttggcc
     7441 tcgcgcgcag atcagttgga agaatttgtc cactacgtga aaggcgagat caccaaggta
     7501 gtcggcaaat aaccctcgag ccacccatga ccaaaatccc ttaacgtgag ttacgcgtcg
     7561 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     7621 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     7681 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     7741 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     7801 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     7861 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     7921 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     7981 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     8041 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     8101 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     8161 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     8221 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     8281 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag c
//
