LOCUS       sequence-306139-        7751 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry clone for CRISPR-iSpyMacCas9 genome editing at NAAR 
            PAMs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7751)
  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 7751)
  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: pYPQ166-iSpyMac
FEATURES             Location/Qualifiers
     source          1..7751
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     127..146
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     380..397
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(556..583)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(675..761)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     810..832
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     824..841
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     825..841
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    857..956
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             978..1043
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1050..1070
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5172..5219
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     protein_bind    5894..6017
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(6034..6053)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6035..6053)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6058..6074)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6058..6074)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             6505..7296
                     /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(7389..7751,1..226)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
       61 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
      121 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
      181 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
      241 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
      301 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
      361 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
      421 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
      481 cgactggaaa gcgggcagtg agcgcaacgc aattaatacg cgtaccgcga gccaggaaga
      541 gtttgtagaa acgcaaaaag gccatccgtc aggatggcct tctgcttagt ttgatgcctg
      601 gcagtttatg gcgggcgtcc tgcccgccac cctccgggcc gttgcttcac aacgttcaaa
      661 tccgctcccg gcggatttgt cctactcagg agagcgttca ccgacaaaca acagataaaa
      721 cgaaaggccc agtcttccga ctgagccttt cgttttattt gatgcctggc agttccctac
      781 tctcgcgtta acgcttgcat ggatgttttc ccagtcacga cgttgtaaaa cgacggccag
      841 tcttaagctc gggccccaaa taatgatttt attttgactg atagtgacct gttcgttgca
      901 acaaattgat gagcaatgct tttttataat gccaactttg tacaaaaaag caggctccga
      961 attcgccctt caccatggat tacaaggacc acgacgggga ttacaaggac cacgacattg
     1021 attacaagga tgatgatgac aagatggctc cgaagaagaa gaggaaggtt ggcatccacg
     1081 gggtgccagc tgctgacaag aagtactcga tcggcctcga tattgggact aactctgttg
     1141 gctgggccgt gatcaccgac gagtacaagg tgccctcaaa gaagttcaag gtcctgggca
     1201 acaccgatcg gcattccatc aagaagaatc tcattggcgc tctcctgttc gacagcggcg
     1261 agacggctga ggctacgcgg ctcaagcgca ccgcccgcag gcggtacacg cgcaggaaga
     1321 atcgcatctg ctacctgcag gagattttct ccaacgagat ggcgaaggtt gacgattctt
     1381 tcttccacag gctggaggag tcattcctcg tggaggagga taagaagcac gagcggcatc
     1441 caatcttcgg caacattgtc gacgaggttg cctaccacga gaagtaccct acgatctacc
     1501 atctgcggaa gaagctcgtg gactccacag ataaggcgga cctccgcctg atctacctcg
     1561 ctctggccca catgattaag ttcaggggcc atttcctgat cgagggggat ctcaacccgg
     1621 acaatagcga tgttgacaag ctgttcatcc agctcgtgca gacgtacaac cagctcttcg
     1681 aggagaaccc cattaatgcg tcaggcgtcg acgcgaaggc tatcctgtcc gctaggctct
     1741 cgaagtctcg gaagctcgag aacctgatcg cccagctgcc gggcgagaag aagaacggcc
     1801 tgttcgggaa tctcattgcg ctcagcctgg ggctcacgcc caacttcaag tcgaatttcg
     1861 atctcgctga ggacgccaag ctgcagctct ccaaggacac atacgacgat gacctggata
     1921 acctcctggc ccagatcggc gatcagtacg cggacctgtt cctcgctgcc aagaatctgt
     1981 cggacgccat cctcctgtct gatattctca gggtgaacac cgagattacg aaggctccgc
     2041 tctcagcctc catgatcaag cgctacgacg agcaccatca ggatctgacc ctcctgaagg
     2101 cgctggtcag gcagcagctc cccgagaagt acaaggagat cttcttcgat cagtcgaaga
     2161 acggctacgc tgggtacatt gacggcgggg cctctcagga ggagttctac aagttcatca
     2221 agccgattct ggagaagatg gacggcacgg aggagctgct ggtgaagctc aagcgcgagg
     2281 acctcctgag gaagcagcgg acattcgata acggcagcat cccacaccag attcatctcg
     2341 gggagctgca cgctatcctg aggaggcagg aggacttcta ccctttcctc aaggataacc
     2401 gcgagaagat cgagaagatt ctgactttca ggatcccgta ctacgtcggc ccactcgcta
     2461 ggggcaactc ccgcttcgct tggatgaccc gcaagtcaga ggagacgatc acgccgtgga
     2521 acttcgagga ggtggtcgac aagggcgcta gcgctcagtc gttcatcgag aggatgacga
     2581 atttcgacaa gaacctgcca aatgagaagg tgctccctaa gcactcgctc ctgtacgagt
     2641 acttcacagt ctacaacgag ctgactaagg tgaagtatgt gaccgagggc atgaggaagc
     2701 cggctttcct gtctggggag cagaagaagg ccatcgtgga cctcctgttc aagaccaacc
     2761 ggaaggtcac ggttaagcag ctcaaggagg actacttcaa gaagattgag tgcttcgatt
     2821 cggtcgagat ctctggcgtt gaggaccgct tcaacgcctc cctggggacc taccacgatc
     2881 tcctgaagat cattaaggat aaggacttcc tggacaacga ggagaatgag gatatcctcg
     2941 aggacattgt gctgacactc actctgttcg aggaccggga gatgatcgag gagcgcctga
     3001 agacttacgc ccatctcttc gatgacaagg tcatgaagca gctcaagagg aggaggtaca
     3061 ccggctgggg gaggctgagc aggaagctca tcaacggcat tcgggacaag cagtccggga
     3121 agacgatcct cgacttcctg aagagcgatg gcttcgcgaa ccgcaatttc atgcagctga
     3181 ttcacgatga cagcctcaca ttcaaggagg atatccagaa ggctcaggtg agcggccagg
     3241 gggactcgct gcacgagcat atcgcgaacc tcgctggctc gccagctatc aagaagggga
     3301 ttctgcagac cgtgaaggtt gtggacgagc tggtgaaggt catgggcagg cacaagcctg
     3361 agaacatcgt cattgagatg gcccgggaga atcagaccac gcagaagggc cagaagaact
     3421 cacgcgagag gatgaagagg atcgaggagg gcattaagga gctggggtcc cagatcctca
     3481 aggagcaccc ggtggagaac acgcagctgc agaatgagaa gctctacctg tactacctcc
     3541 agaatggccg cgatatgtat gtggaccagg agctggatat taacaggctc agcgattacg
     3601 acgtcgatca tatcgttcca cagtcattcc tgaaggatga ctccattgac aacaaggtcc
     3661 tcaccaggtc ggacaagaac cggggcaagt ctgataatgt tccttcagag gaggtcgtta
     3721 agaagatgaa gaactactgg cgccagctcc tgaatgccaa gctgatcacg cagcggaagt
     3781 tcgataacct cacaaaggct gagaggggcg ggctctctga gctggacaag gcgggcttca
     3841 tcaagaggca gctggtcgag acacggcaga tcactaagca cgttgcgcag attctcgact
     3901 cacggatgaa cactaagtac gatgagaatg acaagctgat ccgcgaggtg aaggtcatca
     3961 ccctgaagtc aaagctcgtc tccgacttca ggaaggattt ccagttctac aaggttcggg
     4021 agatcaacaa ttaccaccat gcccatgacg cgtacctgaa cgcggtggtc ggcacagctc
     4081 tgatcaagaa gtacccaaag ctcgagagcg agttcgtgta cggggactac aaggtttacg
     4141 atgtgaggaa gatgatcgcc aagtcggagc aggagattgg caaggctacc gccaagtact
     4201 tcttctactc taacattatg aatttcttca agacagagat cactctggcc aatggcgaga
     4261 tccggaagcg ccccctcatc gagacgaacg gcgagacggg ggagatcgtg tgggacaagg
     4321 gcagggattt cgcgaccgtc aggaaggttc tctccatgcc acaagtgaat atcgtcaaga
     4381 agacagagat acaaacggtt ggtcagaacg ggggtctttt cgatgataac ccaaaatccc
     4441 cgcttgaggt gaccccttca aagctggtgc cactcaaaaa ggaattgaat ccaaaaaagt
     4501 atggcggata ccaaaagcct acaacggcgt atcccgtcct cttgattacg gatacaaagc
     4561 aacttattcc tatatccgtt atgaataaaa aacagttcga gcagaaccct gttaagttcc
     4621 tgcgggatag aggctatcaa caagtgggca agaacgattt tattaagctt cctaaatata
     4681 cgctcgtgga tattggagac ggcataaaaa gactttgggc ttcctcgaaa gaaatacata
     4741 agggcaacca actcgtcgtc tcaaaaaagt ctcagatcct tctttatcat gcgcaccacc
     4801 ttgattcaga cctctcaaac gattatcttc agaaccataa tcagcagttt gacgtgctct
     4861 tcaacgagat aatttcattc tctaaaaagt gcaagctcgg gaaggagcac atacagaaaa
     4921 tcgaaaacgt ctattctaac aaaaagaact cagcctccat agaggagttg gcagaatcat
     4981 tcataaagct cctgggcttt actcagctgg gcgccacatc accgtttaac tttctgggag
     5041 tcaaactcaa tcaaaagcag tataagggca aaaaggacta tattctcccc tgtaccgaag
     5101 gaactctcat caggcaatcc attacagggt tgtacgaaac aagggtcgac ctctcaaaaa
     5161 taggagagga caagcggcca gcggcgacga agaaggcggg gcaggcgaag aagaagaagt
     5221 gagctcagag ctttcgttcg tatcatcggt ttcgacaacg ttcgtcaagt tcaatgcatc
     5281 agtttcattg cgcacacacc agaatcctac tgagtttgag tattatggca ttgggaaaac
     5341 tgtttttctt gtaccatttg ttgtgcttgt aatttactgt gttttttatt cggttttcgc
     5401 tatcgaactg tgaaatggaa atggatggag aagagttaat gaatgatatg gtccttttgt
     5461 tcattctcaa attaatatta tttgtttttt ctcttatttg ttgtgtgttg aatttgaaat
     5521 tataagagat atgcaaacat tttgttttga gtaaaaatgt gtcaaatcgt ggcctctaat
     5581 gaccgaagtt aatatgagga gtaaaacact tgtagttgta ccattatgct tattcactag
     5641 gcaacaaata tattttcaga cctagaaaag ctgcaaatgt tactgaatac aagtatgtcc
     5701 tcttgtgttt tagacattta tgaactttcc tttatgtaat tttccagaat ccttgtcaga
     5761 ttctaatcat tgctttataa ttatagttat actcatggat ttgtagttga gtatgaaaat
     5821 attttttaat gcattttatg acttgccaat tgttccggaa ctctagataa gcttaccgga
     5881 aagggcgaat tcgcaacttt gtatacaaaa gttgaacgag aaacgtaaaa tgatataaat
     5941 atcaatatat taaattagat tttgcataaa aaacagacta cataatactg taaaacacaa
     6001 catatccagt cactatgcca tccagctgat atcccctata gtgagtcgta ttacatggtc
     6061 atagctgttt cctggcagct ctggcccgtg tctcaaaatc tctgatgtta cattgcacaa
     6121 gataaaaata tatcatcatg cctcctctgg accagccagg acagaaatgc ctcgacttcg
     6181 ctgctaccca aggttgccgg gtgacgcaca ccgtggaaac ggatgaaggc acgaacccag
     6241 tggacataag cctgttcggt tcgtaagctg taatgcaagt agcgtatgcg ctcacgcaac
     6301 tggtccagaa ccttgaccga acgcagcggt ggtaacggcg cagtggcggt tttcatggct
     6361 tgttatgact gtttttttgg ggtacagtct atgcctcggg catccaagca gcaagcgcgt
     6421 tacgccgtgg gtcgatgttt gatgttatgg agcagcaacg atgttacgca gcagggcagt
     6481 cgccctaaaa caaagttaaa cattatgagg gaagcggtga tcgccgaagt atcgactcaa
     6541 ctatcagagg tagttggcgt catcgagcgc catctcgaac cgacgttgct ggccgtacat
     6601 ttgtacggct ccgcagtgga tggcggcctg aagccacaca gtgatattga tttgctggtt
     6661 acggtgaccg taaggcttga tgaaacaacg cggcgagctt tgatcaacga ccttttggaa
     6721 acttcggctt cccctggaga gagcgagatt ctccgcgctg tagaagtcac cattgttgtg
     6781 cacgacgaca tcattccgtg gcgttatcca gctaagcgcg aactgcaatt tggagaatgg
     6841 cagcgcaatg acattcttgc aggtatcttc gagccagcca cgatcgacat tgatctggct
     6901 atcttgctga caaaagcaag agaacatagc gttgccttgg taggtccagc ggcggaggaa
     6961 ctctttgatc cggttcctga acaggatcta tttgaggcgc taaatgaaac cttaacgcta
     7021 tggaactcgc cgcccgactg ggctggcgat gagcgaaatg tagtgcttac gttgtcccgc
     7081 atttggtaca gcgcagtaac cggcaaaatc gcgccgaagg atgtcgctgc cgactgggca
     7141 atggagcgcc tgccggccca gtatcagccc gtcatacttg aagctagaca ggcttatctt
     7201 ggacaagaag aagatcgctt ggcctcgcgc gcagatcagt tggaagaatt tgtccactac
     7261 gtgaaaggcg agatcaccaa ggtagtcggc aaataaccct cgagccaccc atgaccaaaa
     7321 tcccttaacg tgagttacgc gtcgttccac tgagcgtcag accccgtaga aaagatcaaa
     7381 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     7441 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     7501 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     7561 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     7621 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     7681 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     7741 cgaacgacct a
//
