LOCUS       sequence-454249-        7073 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  T-DNA vector for SpCas9-D10A based A-to-G base editing for monocot 
            plants; NGG PAM; SpCas9D10A-ABE was driven by ZmUbi1 and the sgRNA 
            was driven by ZmUbi1; Hygromycin for plants selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7073)
  AUTHORS   Cheng Y, Li G, Qi A, Mandlik R, Pan C, Wang D, Ge S, Qi Y
  TITLE     A comprehensive all-in-one CRISPR toolbox for large-scale screens in
            plants.
  JOURNAL   Plant Cell. 2025 Apr 9:koaf081. doi: 10.1093/plcell/koaf081.
  PUBMED    40202185
REFERENCE   2  (bases 1 to 7073)
  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 Cell.
            2025 Apr 9:koaf081. doi: 10.1093/plcell/koaf081."
COMMENT     Sequence Label: pYPQAT36
FEATURES             Location/Qualifiers
     source          1..7073
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1541..2335
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      2422..3010
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2911..2930
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3164..3181
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3196..3336
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3262..3281)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3422..3444
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     rep_origin      3680..3874
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(3940..5013)
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
     CDS             complement(5442..6071)
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
ORIGIN
        1 cgcaagtatt ctactttatg cgactaaaac acgcgacaag aaaacgccag gaaaagggca
       61 gggcggcagc ctgtcgcgta acttaggact tgtgcgacat gtcgttttca gaagacggct
      121 gcactgaacg tcagaagccg actgcactat agcagcggag gggttggatc aaagtacttt
      181 gatcccgagg ggaaccctgt ggttggcatg cacatacaaa tggacgaacg gataaacctt
      241 ttcacgccct tttaaatatc cgattattct aataaacgct cttttctctt agggaaggtg
      301 cgaacaagtc cctgatatga gatcatgttt gtcatctgga gccatagaac agggttcatc
      361 atgagtcatc aacttacctt cgccgacagt gaattcagca gtaagcgccg tcagaccaga
      421 aaagagattt tcttgtcccg catggagcag attctgccat ggcaaaacat ggtggaagtc
      481 atcgagccgt tttaccccaa ggctggtaat ggccggcgac cttatccgct ggaaaccatg
      541 ctacgcattc actgcatgca gcattggtac aacctgagcg atggcgcgat ggaagatgct
      601 ctgtacgaaa tcgcctccat gcgtctgttt gcccggttat ccctggatag cgccttgccg
      661 gaccgcacca ccatcatgaa tttccgccac ctgctggagc agcatcaact ggcccgccaa
      721 ttgttcaaga ccatcaatcg ctggctggcc gaagcaggcg tcatgatgac tcaaggcacc
      781 ttggtcgatg ccaccatcat tgaggcaccc agctcgacca agaacaaaga gcagcaacgc
      841 gatccggaga tgcatcagac caagaaaggc aatcagtggc actttggcat gaaggcccac
      901 attggtgtcg atgccaagag tggcctgacc cacagcctgg tcaccaccgc ggccaacgag
      961 catgacctca atcagctggg taatctgctg catggagagg agcaatttgt ctcagccgat
     1021 gccggctacc aaggggcgcc acagcgcgag gagctggccg aggtggatgt ggactggctg
     1081 atcgccgagc gccccggcaa ggtaagaacc ttgaaacagc atccacgcaa gaacaaaacg
     1141 gccatcaaca tcgaatacat gaaagccagc atccgggcca gggtggagca cccatttcgc
     1201 atcatcaagc gacagttcgg cttcgtgaaa gccagataca aggggttgct gaaaaacgat
     1261 aaccaactgg cgatgttatt cacgctggcc aacctgtttc gggcggacca aatgatacgt
     1321 cagtgggaga gatctcacta aaaactgggg ataacgcctt aaatggcgaa gaaacggtct
     1381 aaataggctg attcaaggca tttacgggag aaaaaatcgg ctcaaacatg aagaaatgaa
     1441 atgactgagt cagccgagaa gaatttcccc gcttattcgc accttcctta gcttcttggg
     1501 gtatctttaa atactgtaga aaagaggaag gaaataataa atggctaaaa tgagaatatc
     1561 accggaattg aaaaaactga tcgaaaaata ccgctgcgta aaagatacgg aaggaatgtc
     1621 tcctgctaag gtatataagc tggtgggaga aaatgaaaac ctatatttaa aaatgacgga
     1681 cagccggtat aaagggacca cctatgatgt ggaacgggaa aaggacatga tgctatggct
     1741 ggaaggaaag ctgcctgttc caaaggtcct gcactttgaa cggcatgatg gctggagcaa
     1801 tctgctcatg agtgaggccg atggcgtcct ttgctcggaa gagtatgaag atgaacaaag
     1861 ccctgaaaag attatcgagc tgtatgcgga gtgcatcagg ctctttcact ccatcgacat
     1921 atcggattgt ccctatacga atagcttaga cagccgctta gccgaattgg attacttact
     1981 gaataacgat ctggccgatg tggattgcga aaactgggaa gaagacactc catttaaaga
     2041 tccgcgcgag ctgtatgatt ttttaaagac ggaaaagccc gaagaggaac ttgtcttttc
     2101 ccacggcgac ctgggagaca gcaacatctt tgtgaaagat ggcaaagtaa gtggctttat
     2161 tgatcttggg agaagcggca gggcggacaa gtggtatgac attgccttct gcgtccggtc
     2221 gatcagggag gatatcgggg aagaacagta tgtcgagcta ttttttgact tactggggat
     2281 caagcctgat tgggagaaaa taaaatatta tattttactg gatgaattgt tttagtacct
     2341 agaatgcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     2401 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     2461 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     2521 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     2581 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     2641 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     2701 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     2761 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     2821 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     2881 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     2941 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     3001 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     3061 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     3121 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     3181 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     3241 ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     3301 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     3361 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     3421 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagggt
     3481 gccttgatgt gggcgccggc ggtcgagtgg cgacggcgcg gcttgtccgc gccctggtag
     3541 attgcctggc cgtaggccag ccatttttga gcggccagcg gccgcgatag gccgacgcga
     3601 agcggcgggg cgtagggagc gcagcgaccg aagggtaggc gctttttgca gctcttcggc
     3661 tgtgcgctgg ccagacagtt atgcacaggc caggcgggtt ttaagagttt taataagttt
     3721 taaagagttt taggcggaaa aatcgccttt tttctctttt atatcagtca cttacatgtg
     3781 tgaccggttc ccaatgtacg gctttgggtt cccaatgtac gggttccggt tcccaatgta
     3841 cggctttggg ttcccaatgt acgtgctatc cacaggaaac agaccttttc gacctttttc
     3901 ccctgctagg gcaatttgcc ctagcatctg ctccgtacat taggaaccgg cggatgcttc
     3961 gccctcgatc aggttgcggt agcgcatgac taggatcggg ccagcctgcc ccgcctcctc
     4021 cttcaaatcg tactccggca ggtcatttga cccgatcagc ttgcgcacgg tgaaacagaa
     4081 cttcttgaac tctccggcgc tgccactgcg ttcgtagatc gtcttgaaca accatctggc
     4141 ttctgccttg cctgcggcgc ggcgtgccag gcggtagaga aaacggccga tgccgggatc
     4201 gatcaaaaag taatcggggt gaaccgtcag cacgtccggg ttcttgcctt ctgtgatctc
     4261 gcggtacatc caatcagcta gctcgatctc gatgtactcc ggccgcccgg tttcgctctt
     4321 tacgatcttg tagcggctaa tcaaggcttc accctcggat accgtcacca ggcggccgtt
     4381 cttggccttc ttcgtacgct gcatggcaac gtgcgtggtg tttaaccgaa tgcaggtttc
     4441 taccaggtcg tctttctgct ttccgccatc ggctcgccgg cagaacttga gtacgtccgc
     4501 aacgtgtgga cggaacacgc ggccgggctt gtctcccttc ccttcccggt atcggttcat
     4561 ggattcggtt agatgggaaa ccgccatcag taccaggtcg taatcccaca cactggccat
     4621 gccggccggc cctgcggaaa cctctacgtg cccgtctgga agctcgtagc ggatcacctc
     4681 gccagctcgt cggtcacgct tcgacagacg gaaaacggcc acgtccatga tgctgcgact
     4741 atcgcgggtg cccacgtcat agagcatcgg aacgaaaaaa tctggttgct cgtcgccctt
     4801 gggcggcttc ctaatcgacg gcgcaccggc tgccggcggt tgccgggatt ctttgcggat
     4861 tcgatcagcg gccgcttgcc acgattcacc ggggcgtgct tctgcctcga tgcgttgccg
     4921 ctgggcggcc tgcgcggcct tcaacttctc caccaggtca tcacccagcg ccgcgccgat
     4981 ttgtaccggg ccggatggtt tgcgaccgct cacgccgatt cctcgggctt gggggttcca
     5041 gtgccattgc agggccggca ggcaacccag ccgcttacgc ctggccaacc gcccgttcct
     5101 ccacacatgg ggcattccac ggcgtcggtg cctggttgtt cttgattttc catgccgcct
     5161 cctttagccg ctaaaattca tctactcatt tattcatttg ctcatttact ctggtagctg
     5221 cgcgatgtat tcagatagca gctcggtaat ggtcttgcct tggcgtaccg cgtacatctt
     5281 cagcttggtg tgatcctccg ccggcaactg aaagttgacc cgcttcatgg ctggcgtgtc
     5341 tgccaggctg gccaacgttg cagccttgct gctgcgtgcg ctcggacggc cggcacttag
     5401 cgtgtttgtg cttttgctca ttttctcttt acctcattaa ctcaaatgag ttttgattta
     5461 atttcagcgg ccagcgcctg gacctcgcgg gcagcgtcgc cctcgggttc tgattcaaga
     5521 acggttgtgc cggcggcggc agtgcctggg tagctcacgc gctgcgtgat acgggactca
     5581 agaatgggca gctcgtaccc ggccagcgcc tcggcaacct caccgccgat gcgcgtgcct
     5641 ttgatcgccc gcgacacgac aaaggccgct tgtagccttc catccgtgac ctcaatgcgc
     5701 tgcttaacca gctccaccag gtcggcggtg gcccatatgt cgtaagggct tggctgcacc
     5761 ggaatcagca cgaagtcggc tgccttgatc gcggacacag ccaagtccgc cgcctggggc
     5821 gctccgtcga tcactacgaa gtcgcgccgg ccgatggcct tcacgtcgcg gtcaatcgtc
     5881 gggcggtcga tgccgacaac ggttagcggt tgatcttccc gcacggccgc ccaatcgcgg
     5941 gcactgccct ggggatcgga atcgactaac agaacatcgg ccccggcgag ttgcagggcg
     6001 cgggctagat gggttgcgat ggtcgtcttg cctgacccgc ctttctggtt aagtacagcg
     6061 ataaccttca tgcgttcccc ttgcgtattt gtttatttac tcatcgcatc atatacgcag
     6121 cgaccgcatg acgcaagctg ttttactcaa atacacatca cctttttaga cggcggcgct
     6181 cggtttcttc agcggccaag ctggccggcc aggccgccag cttggcatca gacaaaccgg
     6241 ccaggatttc atgcagccgc acggttgaga cgtgcgcggg cggctcgaac acgtacccgg
     6301 ccgcgatcat ctccgcctcg atctcttcgg taatgaaaaa cggttcgtcc tggccgtcct
     6361 ggtgcggttt catgcttgtt cctcttggcg ttcattctcg gcggccgcca gggcgtcggc
     6421 ctcggtcaat gcgtcctagg caccgcgccg cctggcctcg gtgggcgtca cttcctcgct
     6481 gcgctcaagt gcgcggtaca gggtcgagcg atgcacgcca agcagtgcag ccgcctcttt
     6541 cacggtgcgg ccttcctggt cgatcagctc gcgggcgtgc gcgatctgtg ccggggtgag
     6601 ggtagggcgg gggccaaact tcacgcctcg ggccttggcg gcctcgcgcc cgctccgggt
     6661 gcggtcgatg attagggaac gctcgaactc ggcaatgccg gcgaacacgg tcaacaccat
     6721 gcggccggcc ggcgtggtgg tgtcggccca cggctctgcc aggctacgca ggcccgcgcc
     6781 ggcctcctgg atgcgctcgg caatgtccag taggtcgcgg gtgctgcggg ccaggcggtc
     6841 tagcctggtc actgtcacaa cgtcgccagg gcgtaggtgg tcaagcatcc tggccagctc
     6901 cgggcggtcg cgcctggtgc cggtgatctt ctcggaaaac agcttggtgc agccggccgc
     6961 gtgcagttcg gcccgttggt tggtcaagtc ctggtcgtcg gtgctgacgc gggcatagcc
     7021 cagcaggcca gcggcggcgc tcttgttcat ggcgtaatgt ctccggttct agt
//
