LOCUS       sequence-306140-        8817 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry clone for CRISPR-iSpyMacCas9 PmCDA1 C-T base editing 
            at NAAR PAMs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8817)
  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 8817)
  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: pYPQ266B
FEATURES             Location/Qualifiers
     source          1..8817
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     118..137
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     371..388
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(547..574)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(666..752)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     801..823
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     815..832
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     816..832
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    848..947
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             969..1034
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1041..1061
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5163..5210
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5427..5492
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             6126..6146
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6153..6404
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHT
                     AYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             6405..6458
                     /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      6654..6906
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    6951..7074
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(7091..7110)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7092..7110)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7115..7131)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7115..7131)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             7562..8353
                     /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      join(8446..8817,1..217)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
       61 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
      121 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
      181 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
      241 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
      301 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
      361 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
      421 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
      481 agcgggcagt gagcgcaacg caattaatac gcgtaccgcg agccaggaag agtttgtaga
      541 aacgcaaaaa ggccatccgt caggatggcc ttctgcttag tttgatgcct ggcagtttat
      601 ggcgggcgtc ctgcccgcca ccctccgggc cgttgcttca caacgttcaa atccgctccc
      661 ggcggatttg tcctactcag gagagcgttc accgacaaac aacagataaa acgaaaggcc
      721 cagtcttccg actgagcctt tcgttttatt tgatgcctgg cagttcccta ctctcgcgtt
      781 aacgcttgca tggatgtttt cccagtcacg acgttgtaaa acgacggcca gtcttaagct
      841 cgggccccaa ataatgattt tattttgact gatagtgacc tgttcgttgc aacaaattga
      901 tgagcaatgc ttttttataa tgccaacttt gtacaaaaaa gcaggctccg aattcgccct
      961 tcaccatgga ttacaaggac cacgacgggg attacaagga ccacgacatt gattacaagg
     1021 atgatgatga caagatggct ccgaagaaga agaggaaggt tggcatccac ggggtgccag
     1081 ctgctgacaa gaagtactcg atcggcctcg ctattgggac taactctgtt ggctgggccg
     1141 tgatcaccga cgagtacaag gtgccctcaa agaagttcaa ggtcctgggc aacaccgatc
     1201 ggcattccat caagaagaat ctcattggcg ctctcctgtt cgacagcggc gagacggctg
     1261 aggctacgcg gctcaagcgc accgcccgca ggcggtacac gcgcaggaag aatcgcatct
     1321 gctacctgca ggagattttc tccaacgaga tggcgaaggt tgacgattct ttcttccaca
     1381 ggctggagga gtcattcctc gtggaggagg ataagaagca cgagcggcat ccaatcttcg
     1441 gcaacattgt cgacgaggtt gcctaccacg agaagtaccc tacgatctac catctgcgga
     1501 agaagctcgt ggactccaca gataaggcgg acctccgcct gatctacctc gctctggccc
     1561 acatgattaa gttcaggggc catttcctga tcgaggggga tctcaacccg gacaatagcg
     1621 atgttgacaa gctgttcatc cagctcgtgc agacgtacaa ccagctcttc gaggagaacc
     1681 ccattaatgc gtcaggcgtc gacgcgaagg ctatcctgtc cgctaggctc tcgaagtctc
     1741 ggaagctcga gaacctgatc gcccagctgc cgggcgagaa gaagaacggc ctgttcggga
     1801 atctcattgc gctcagcctg gggctcacgc ccaacttcaa gtcgaatttc gatctcgctg
     1861 aggacgccaa gctgcagctc tccaaggaca catacgacga tgacctggat aacctcctgg
     1921 cccagatcgg cgatcagtac gcggacctgt tcctcgctgc caagaatctg tcggacgcca
     1981 tcctcctgtc tgatattctc agggtgaaca ccgagattac gaaggctccg ctctcagcct
     2041 ccatgatcaa gcgctacgac gagcaccatc aggatctgac cctcctgaag gcgctggtca
     2101 ggcagcagct ccccgagaag tacaaggaga tcttcttcga tcagtcgaag aacggctacg
     2161 ctgggtacat tgacggcggg gcctctcagg aggagttcta caagttcatc aagccgattc
     2221 tggagaagat ggacggcacg gaggagctgc tggtgaagct caagcgcgag gacctcctga
     2281 ggaagcagcg gacattcgat aacggcagca tcccacacca gattcatctc ggggagctgc
     2341 acgctatcct gaggaggcag gaggacttct accctttcct caaggataac cgcgagaaga
     2401 tcgagaagat tctgactttc aggatcccgt actacgtcgg cccactcgct aggggcaact
     2461 cccgcttcgc ttggatgacc cgcaagtcag aggagacgat cacgccgtgg aacttcgagg
     2521 aggtggtcga caagggcgct agcgctcagt cgttcatcga gaggatgacg aatttcgaca
     2581 agaacctgcc aaatgagaag gtgctcccta agcactcgct cctgtacgag tacttcacag
     2641 tctacaacga gctgactaag gtgaagtatg tgaccgaggg catgaggaag ccggctttcc
     2701 tgtctgggga gcagaagaag gccatcgtgg acctcctgtt caagaccaac cggaaggtca
     2761 cggttaagca gctcaaggag gactacttca agaagattga gtgcttcgat tcggtcgaga
     2821 tctctggcgt tgaggaccgc ttcaacgcct ccctggggac ctaccacgat ctcctgaaga
     2881 tcattaagga taaggacttc ctggacaacg aggagaatga ggatatcctc gaggacattg
     2941 tgctgacact cactctgttc gaggaccggg agatgatcga ggagcgcctg aagacttacg
     3001 cccatctctt cgatgacaag gtcatgaagc agctcaagag gaggaggtac accggctggg
     3061 ggaggctgag caggaagctc atcaacggca ttcgggacaa gcagtccggg aagacgatcc
     3121 tcgacttcct gaagagcgat ggcttcgcga accgcaattt catgcagctg attcacgatg
     3181 acagcctcac attcaaggag gatatccaga aggctcaggt gagcggccag ggggactcgc
     3241 tgcacgagca tatcgcgaac ctcgctggct cgccagctat caagaagggg attctgcaga
     3301 ccgtgaaggt tgtggacgag ctggtgaagg tcatgggcag gcacaagcct gagaacatcg
     3361 tcattgagat ggcccgggag aatcagacca cgcagaaggg ccagaagaac tcacgcgaga
     3421 ggatgaagag gatcgaggag ggcattaagg agctggggtc ccagatcctc aaggagcacc
     3481 cggtggagaa cacgcagctg cagaatgaga agctctacct gtactacctc cagaatggcc
     3541 gcgatatgta tgtggaccag gagctggata ttaacaggct cagcgattac gacgtcgatc
     3601 atatcgttcc acagtcattc ctgaaggatg actccattga caacaaggtc ctcaccaggt
     3661 cggacaagaa ccggggcaag tctgataatg ttccttcaga ggaggtcgtt aagaagatga
     3721 agaactactg gcgccagctc ctgaatgcca agctgatcac gcagcggaag ttcgataacc
     3781 tcacaaaggc tgagaggggc gggctctctg agctggacaa ggcgggcttc atcaagaggc
     3841 agctggtcga gacacggcag atcactaagc acgttgcgca gattctcgac tcacggatga
     3901 acactaagta cgatgagaat gacaagctga tccgcgaggt gaaggtcatc accctgaagt
     3961 caaagctcgt ctccgacttc aggaaggatt tccagttcta caaggttcgg gagatcaaca
     4021 attaccacca tgcccatgac gcgtacctga acgcggtggt cggcacagct ctgatcaaga
     4081 agtacccaaa gctcgagagc gagttcgtgt acggggacta caaggtttac gatgtgagga
     4141 agatgatcgc caagtcggag caggagattg gcaaggctac cgccaagtac ttcttctact
     4201 ctaacattat gaatttcttc aagacagaga tcactctggc caatggcgag atccggaagc
     4261 gccccctcat cgagacgaac ggcgagacgg gggagatcgt gtgggacaag ggcagggatt
     4321 tcgcgaccgt caggaaggtt ctctccatgc cacaagtgaa tatcgtcaag aagacagaga
     4381 tacaaacggt tggtcagaac gggggtcttt tcgatgataa cccaaaatcc ccgcttgagg
     4441 tgaccccttc aaagctggtg ccactcaaaa aggaattgaa tccaaaaaag tatggcggat
     4501 accaaaagcc tacaacggcg tatcccgtcc tcttgattac ggatacaaag caacttattc
     4561 ctatatccgt tatgaataaa aaacagttcg agcagaaccc tgttaagttc ctgcgggata
     4621 gaggctatca acaagtgggc aagaacgatt ttattaagct tcctaaatat acgctcgtgg
     4681 atattggaga cggcataaaa agactttggg cttcctcgaa agaaatacat aagggcaacc
     4741 aactcgtcgt ctcaaaaaag tctcagatcc ttctttatca tgcgcaccac cttgattcag
     4801 acctctcaaa cgattatctt cagaaccata atcagcagtt tgacgtgctc ttcaacgaga
     4861 taatttcatt ctctaaaaag tgcaagctcg ggaaggagca catacagaaa atcgaaaacg
     4921 tctattctaa caaaaagaac tcagcctcca tagaggagtt ggcagaatca ttcataaagc
     4981 tcctgggctt tactcagctg ggcgccacat caccgtttaa ctttctggga gtcaaactca
     5041 atcaaaagca gtataagggc aaaaaggact atattctccc ctgtaccgaa ggaactctca
     5101 tcaggcaatc cattacaggg ttgtacgaaa caagggtcga cctctcaaaa ataggagagg
     5161 acaagcggcc agcggcgacg aagaaggcgg ggcaggcgaa gaagaagaag ggagacgacg
     5221 gaggtggcgg tacaggaggg ggtgggtccg ctgagtatgt cagggcgttg ttcgacttca
     5281 atggaaacga cgaggaagat ctgcctttta aaaagggaga tattctcagg atcagagata
     5341 agccggaaga acaatggtgg aacgctgaag actctgaagg taagagaggt atgattcttg
     5401 tcccctacgt cgagaagtat tcgggtgact ataaagacca cgatggagat tataaggacc
     5461 acgatataga ttataaggat gatgatgata agagcggaat gaccgatgca gagtacgtca
     5521 ggattcatga gaaacttgac atctacacgt ttaagaaaca gtttttcaac aacaaaaaat
     5581 ctgttagtca ccgctgttac gtgctgttcg aattgaaacg cagaggtgag aggagagcct
     5641 gcttttgggg ctatgccgtc aacaagccgc aaagcggcac agaaaggggc attcacgcgg
     5701 agatatttag cattagaaag gtcgaggaat accttcggga taatcccggg caattcacta
     5761 tcaattggta ctcttcatgg tccccgtgtg cagattgcgc tgaaaagata ctggagtggt
     5821 ataatcaaga actcagagga aacggtcaca ccctcaagat ttgggcttgc aagctttact
     5881 acgagaaaaa tgcaaggaac cagatcggcc tctggaactt gcgcgacaac ggcgtggggt
     5941 tgaatgtgat ggtctcggag cattaccagt gctgccggaa gatattcatt cagtcgtcac
     6001 ataatcaatt gaacgagaat aggtggctcg aaaaaaccct gaagcgggcc gagaagtgga
     6061 ggagtgaact ctcgataatg atccaggtta aaatactgca tactaccaaa tctccggcgg
     6121 tgggaccgaa gaagaagcgc aaggtgggga ccatgactaa tctctcagat ataatcgaga
     6181 aggaaacagg aaagcaactg gtcatccaag aatcgatttt gatgcttccc gaagaagtcg
     6241 aagaagttat aggaaataag cccgagtctg acatactggt tcacacagcg tacgatgaaa
     6301 gtacggacga gaatgtcatg ttgctgacat cggacgcacc tgaatacaag ccttgggctc
     6361 tggtcataca agatagtaac ggagaaaata agattaaaat gcttgaagga aggggctccc
     6421 tgctgacatg tggagatgtg gaagagaatc caggaccatc caggatgata gagcaagatg
     6481 gttgtaccca agtgctgcgg cccctcggat ggcgggggta cagcgccatg actggccata
     6541 atagacagtc cgctgctctc atgcctccat gcagcggatg tcagaggagg ggagctaggt
     6601 tcttcctgtc tagacctacc tgccccgtgc cctagtggcg gccgccgacg tccgatcgtt
     6661 caaacatttg gcaataaagt ttcttaagat tgaatcctgt tgccggtctt gcgatgatta
     6721 tcatataatt tctgttgaat tacgttaagc atgtaataat taacatgtaa tgcatgacgt
     6781 tatttatgag atgggttttt atgattagag tcccgcaatt atacatttaa tacgcgatag
     6841 aaaacaaaat atagcgcgca aactaggata aattatcgcg cgcggtgtca tctatgttac
     6901 tagatcggga attgatcccc cctcgacagc ttccggaaag ggcgaattcg caactttgta
     6961 tacaaaagtt gaacgagaaa cgtaaaatga tataaatatc aatatattaa attagatttt
     7021 gcataaaaaa cagactacat aatactgtaa aacacaacat atccagtcac tatgccatcc
     7081 agctgatatc ccctatagtg agtcgtatta catggtcata gctgtttcct ggcagctctg
     7141 gcccgtgtct caaaatctct gatgttacat tgcacaagat aaaaatatat catcatgcct
     7201 cctctggacc agccaggaca gaaatgcctc gacttcgctg ctacccaagg ttgccgggtg
     7261 acgcacaccg tggaaacgga tgaaggcacg aacccagtgg acataagcct gttcggttcg
     7321 taagctgtaa tgcaagtagc gtatgcgctc acgcaactgg tccagaacct tgaccgaacg
     7381 cagcggtggt aacggcgcag tggcggtttt catggcttgt tatgactgtt tttttggggt
     7441 acagtctatg cctcgggcat ccaagcagca agcgcgttac gccgtgggtc gatgtttgat
     7501 gttatggagc agcaacgatg ttacgcagca gggcagtcgc cctaaaacaa agttaaacat
     7561 tatgagggaa gcggtgatcg ccgaagtatc gactcaacta tcagaggtag ttggcgtcat
     7621 cgagcgccat ctcgaaccga cgttgctggc cgtacatttg tacggctccg cagtggatgg
     7681 cggcctgaag ccacacagtg atattgattt gctggttacg gtgaccgtaa ggcttgatga
     7741 aacaacgcgg cgagctttga tcaacgacct tttggaaact tcggcttccc ctggagagag
     7801 cgagattctc cgcgctgtag aagtcaccat tgttgtgcac gacgacatca ttccgtggcg
     7861 ttatccagct aagcgcgaac tgcaatttgg agaatggcag cgcaatgaca ttcttgcagg
     7921 tatcttcgag ccagccacga tcgacattga tctggctatc ttgctgacaa aagcaagaga
     7981 acatagcgtt gccttggtag gtccagcggc ggaggaactc tttgatccgg ttcctgaaca
     8041 ggatctattt gaggcgctaa atgaaacctt aacgctatgg aactcgccgc ccgactgggc
     8101 tggcgatgag cgaaatgtag tgcttacgtt gtcccgcatt tggtacagcg cagtaaccgg
     8161 caaaatcgcg ccgaaggatg tcgctgccga ctgggcaatg gagcgcctgc cggcccagta
     8221 tcagcccgtc atacttgaag ctagacaggc ttatcttgga caagaagaag atcgcttggc
     8281 ctcgcgcgca gatcagttgg aagaatttgt ccactacgtg aaaggcgaga tcaccaaggt
     8341 agtcggcaaa taaccctcga gccacccatg accaaaatcc cttaacgtga gttacgcgtc
     8401 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     8461 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     8521 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     8581 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     8641 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     8701 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     8761 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaact
//
