LOCUS       sequence-252953-       10031 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level1 ZmUBIp::StCas9 (Arabidopsis codon optimized):NOSt.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10031)
  AUTHORS   Hahn F, Korolev A, Sanjurjo Loures L, Nekrasov V
  TITLE     A modular cloning toolkit for genome editing in plants.
  JOURNAL   BMC Plant Biol. 2020 Apr 23;20(1):179. doi: 
            10.1186/s12870-020-02388-2.
  PUBMED    32326901
REFERENCE   2  (bases 1 to 10031)
  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: "BMC Plant 
            Biol."; date: "2020-04-23"; volume: "20(1):179. doi"; pages: " 
            10.1186/s12870-020-02388-2"
COMMENT     Sequence Label: pFH59
FEATURES             Location/Qualifiers
     source          1..10031
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             5398..5418
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      5434..5686
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     misc_feature    5734..5758
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(5914..7062)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     rep_origin      complement(7398..7986)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7478..7497)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8157..8948,8949..9017))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     8780..8799
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9018..9122)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      9209..9840
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    9932..9956
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 tctgtgaaga caagcaagaa ttcaagcttg gaggtgcagc gtgacccggt cgtgcccctc
       61 tctagagata atgagcattg catgtctaag ttataaaaaa ttaccacata ttttttttgt
      121 cacacttgtt tgaagtgcag tttatctatc tttatacata tatttaaact ttactctacg
      181 aataatataa tctatagtac tacaataata tcagtgtttt agagaatcat ataaatgaac
      241 agttagacat ggtctaaagg acaattgagt attttgacaa caggactcta cagttttatc
      301 tttttagtgt gcatgtgttc tccttttttt ttgcaaatag cttcacctat ataatacttc
      361 atccatttta ttagtacatc catttagggt ttagggttaa tggtttttat agactaattt
      421 ttttagtaca tctattttat tctattttag cctctaaatt aagaaaacta aaactctatt
      481 ttagtttttt tatttaataa tttagatata aaatagaata aaataaagtg actaaaaatt
      541 aaacaaatac cctttaagaa attaaaaaaa ctaaggaaac atttttcttg tttcgagtag
      601 ataatgccag cctgttaaac gccgtcgacg agtctaacgg acaccaacca gcgaaccagc
      661 agcgtcgcgt cgggccaagc gaagcagacg gcacggcatc tctgtcgctg cctctggacc
      721 cctctcgaga gttccgctcc accgttggac ttgctccgct gtcggcatcc agaaattgcg
      781 tggcggagcg gcagacgtga gccggcacgg caggcggcct cctcctcctc tcacggcacg
      841 gcagctacgg gggattcctt tcccaccgct ccttcgcttt cccttcctcg cccgccgtaa
      901 taaatagaca ccccctccac accctctttc cccaacctcg tgttgttcgg agcgcacaca
      961 cacacaacca gatctccccc aaatccaccc gtcggcacct ccgcttcaag gtacgccgct
     1021 cgtcctcccc ccccccccct ctctaccttc tctagatcgg cgttccggtc catggttagg
     1081 gcccggtagt tctacttctg ttcatgtttg tgttagatcc gtgtttgtgt tagatccgtg
     1141 ctgctagcgt tcgtacacgg atgcgacctg tacgtcagac acgttctgat tgctaacttg
     1201 ccagtgtttc tctttgggga atcctgggat ggctctagcc gttccgcaga cgggatcgat
     1261 ttcatgattt tttttgtttc gttgcatagg gtttggtttg cccttttcct ttatttcaat
     1321 atatgccgtg cacttgtttg tcgggtcatc ttttcatgct tttttttgtc ttggttgtga
     1381 tgatgtggtc tggttgggcg gtcgttctag atcggagtag aattctgttt caaactacct
     1441 ggtggattta ttaattttgg atctgtatgt gtgtgccata catattcata gttacgaatt
     1501 gaagatgatg gatggaaata tcgatctagg ataggtatac atgttgatgc gggttttact
     1561 gatgcatata cagagatgct ttttgttcgc ttggttgtga tgatgtggtg tggttgggcg
     1621 gtcgttcatt cgttctagat cggagtagaa tactgtttca aactacctgg tgtatttatt
     1681 aattttggaa ctgtatgtgt gtgtcataca tcttcatagt tacgagttta agatggatgg
     1741 aaatatcgat ctaggatagg tatacatgtt gatgtgggtt ttactgatgc atatacatga
     1801 tggcatatgc agcatctatt catatgctct aaccttgagt acctatctat tataataaac
     1861 aagtatgttt tataattatt ttgatcttga tatacttgga tgatggcata tgcagcagct
     1921 atatgtggat ttttttagcc ctgccttcat acgctattta tttgcttggt actgtttctt
     1981 ttgtcgatgc tcaccctgtt gtttggtgtt acttctgcag gaatgtctga tctcgtgctc
     2041 ggactcgata tcggaatcgg atctgttgga gtgggaatcc tcaacaaggt taccggtgag
     2101 atcatccaca agaactctag gatcttccct gctgctcagg ctgagaacaa cctcgtgaga
     2161 agaactaaca ggcagggtag aaggctcgct agaagaaaga agcacagaag ggttaggctc
     2221 aacagactct tcgaggaatc tggactcatc accgatttca ccaagatctc tatcaacctc
     2281 aacccttacc agctcagggt gaagggactt accgatgagc tttctaacga ggaactcttc
     2341 attgctctca agaacatggt gaagcacagg ggtatcagtt acctcgatga tgcttctgat
     2401 gatggaaact cttctgtggg agattacgct cagatcgtga aagagaactc taagcagctc
     2461 gagactaaga cccctggaca aatccagctt gagagatacc agacttacgg acagcttagg
     2521 ggagatttca ccgttgagaa ggatggaaag aaacacaggc tcatcaacgt gttccctacc
     2581 tctgcttaca gatctgaggc tctcagaatc ctccagaccc agcaagaatt caaccctcag
     2641 atcaccgatg agttcatcaa cagatacctc gagatcctca ctggtaagag gaagtactat
     2701 cacggacctg gaaacgagaa gtctaggacc gattacggaa ggtacagaac ctctggtgag
     2761 actctcgata acatcttcgg tatcctcatc ggaaagtgca cattctaccc agatgaattc
     2821 agggctgcta aggcttctta caccgctcaa gagttcaacc tcctcaacga tctcaacaac
     2881 ctcactgtgc ctaccgagac aaagaagctc tctaaagagc agaagaacca gatcatcaac
     2941 tacgtgaaga acgagaaggc tatgggacct gctaagcttt tcaagtatat cgctaagctc
     3001 ctctcttgtg atgtggctga tatcaaggga tacaggatcg ataagtctgg aaaggctgag
     3061 atccacacct tcgaggctta cagaaagatg aaaaccctcg aaaccttgga tatcgagcag
     3121 atggataggg aaaccctcga taagctcgct tacgtgctca ctctcaacac tgagagagag
     3181 ggaatccaag aggctctcga acatgagttc gctgatggat ctttctcaca aaagcaagtg
     3241 gatgagcttg tgcagttcag aaaggctaac tcttctatct tcggaaaggg atggcacaac
     3301 ttctctgtga agcttatgat ggaacttatc cctgagcttt acgagacttc tgaggaacag
     3361 atgaccatcc tcaccaggct cggaaagcaa aagaccacct cttcatctaa caagaccaag
     3421 tatattgatg agaagctcct caccgaagag atctacaacc ctgtggttgc taagtctgtg
     3481 aggcaggcta tcaagatcgt taacgctgct atcaaagaat acggtgattt tgataacatc
     3541 gtgatcgaga tggcaaggga aacaaacgag gatgatgaga aaaaggctat ccaaaagatc
     3601 cagaaagcta acaaggatga gaaggatgct gctatgctca aggctgctaa ccagtacaat
     3661 ggtaaggctg agcttcctca ctctgtgttc catggtcata agcagcttgc taccaagatc
     3721 agactctggc atcaacaggg tgagagatgc ctctacactg gaaagaccat ctctatccat
     3781 gatctcatca acaactctaa ccagttcgaa gtggatcaca tcctcccact ctcaatcacc
     3841 ttcgatgatt ctctcgcaaa caaggtgctc gtgtacgcta ccgctaatca agagaaggga
     3901 cagaggactc cataccaggc actcgattct atggatgatg catggtcatt cagggaactc
     3961 aaggctttcg tgagagagtc taagaccctc agtaacaaga aaaaagagta cctcctcact
     4021 gaggaagata tctctaagtt cgatgtgagg aagaagttca ttgagaggaa cctcgtggat
     4081 accaggtacg cttctagagt tgtgctcaac gctctccaag agcatttcag ggctcacaag
     4141 atcgatacaa aggtgtcagt ggtgagggga cagttcactt ctcagcttag aaggcactgg
     4201 ggaatcgaaa agaccaggga tacttaccat caccacgctg tggatgctct tattatcgct
     4261 gcttcatctc agctcaacct ctggaagaag cagaaaaaca ccctcgtgtc ttactctgag
     4321 gatcagctcc tcgatattga gactggtgag cttatctcag atgatgagta caaagagtca
     4381 gttttcaagg caccatacca gcatttcgtt gataccctca agtctaaaga gttcgaggat
     4441 tctatcctct tcagttacca agtggattct aagttcaata gaaagatctc tgatgctacc
     4501 atctacgcta ccaggcaagc taaggtggga aaggataagg ctgatgagac atacgtgctc
     4561 ggtaagatca aggatatcta cacccaggat ggatacgatg ctttcatgaa gatctataag
     4621 aaggataagt ctaagttcct catgtacagg cacgatccac agacattcga gaaggtgatc
     4681 gagcctatcc tcgagaacta ccctaacaag cagatcaacg ataagggaaa agaagtgcct
     4741 tgcaacccat tcctcaagta caaagaagaa cacggttaca tcagaaagta ctctaagaag
     4801 ggaaacggac ctgagatcaa gtctctcaag tactacgatt ctaagctcgg aaaccacatc
     4861 gatatcaccc ctaagaattc taacaacaag gtggtgctcc agtctgtgtc tccttggagg
     4921 gctgatgtgt acttcaacaa gactaccgga aagtacgaga tccttggact caagtacgct
     4981 gatctccagt tcgataaggg tactggaacc tacaagatct cacaagaaaa gtacaacgat
     5041 atcaaaaaga aagagggtgt tgattctgat tcagagttca agttcaccct ctacaagaac
     5101 gatttgctcc tcgtgaagga tactgagaca aaagagcagc agcttttcag attcctttct
     5161 aggaccatgc ctaagcagaa gcactacgtt gagcttaagc cttacgataa gcaaaagttc
     5221 gagggtggtg aggcactcat caaggttctc ggaaacgttg caaactctgg acagtgcaag
     5281 aagggactcg gaaagtctaa catctcaatc tacaaggtga ggaccgatgt gcttggaaac
     5341 cagcacatca tcaagaatga gggtgataag cctaagctcg atttctcaag ggctgatcct
     5401 aagaagaaga ggaaggtttg agcttgtcaa gcagatcgtt caaacatttg gcaataaagt
     5461 ttcttaagat tgaatcctgt tgccggtctt gcgatgatta tcatataatt tctgttgaat
     5521 tacgttaagc atgtaataat taacatgtaa tgcatgacgt tatttatgag atgggttttt
     5581 atgattagag tcccgcaatt atacatttaa tacgcgatag aaaacaaaat atagcgcgca
     5641 aactaggata aattatcgcg cgcggtgtca tctatgttac tagatcgacg ctactattgt
     5701 cttctgcacg aagtggttta aactatcagt gtttgacagg atatattggc gggtaaacct
     5761 aagagaaaag agcgtttatt agaataatcg gatatttaaa agggcgtgaa aaggtttatc
     5821 cgttcgtcca tttgtatgtg catgccaacc acagggttcc ccagatcagg cgctggctgc
     5881 tgaaccccca gccggaactg accccacaag gccctagcgt ttgcaatgca ccaggtcatc
     5941 attgacccag gcgtgttcca ccaggccgct gcctcgcaac tcttcgcagg cttcgccgac
     6001 ctgctcgcgc cacttcttca cgcgggtgga atccgatccg cacatgaggc ggaaggtttc
     6061 cagcttgagc gggtacggct cccggtgcga gctgaaatag tcgaacatcc gtcgggccgt
     6121 cggcgacagc ttgcggtact tctcccatat gaatttcgtg tagtggtcgc cagcaaacag
     6181 cacgacgatt tcctcgtcga tcaggacctg gcaacgggac gttttcttgc cacggtccag
     6241 gacgcggaag cggtgcagca gcgacaccga ttccaggtgc ccaacgcggt cggacgtgaa
     6301 gcccatcgcc gtcgcctgta ggcgcgacag gcattcctcg gccttcgtgt aataccggcc
     6361 attgatcgac cagcccaggt cctggcaaag ctcgtagaac gtgaaggtga tcggctcgcc
     6421 gataggggtg cgcttcgcgt actccaacac ctgctgccac accagttcgt catcgtcggc
     6481 ccgcagctcg acgccggtgt aggtgatctt cacgtccttg ttgacgtgga aaatgacctt
     6541 gttttgcagc gcctcgcgcg ggattttctt gttgcgcgtg gtgaacaggg cagagcgggc
     6601 cgtgtcgttt ggcatcgctc gcatcgtgtc cggccacggc gcaatatcga acaaggaaag
     6661 ctgcatttcc ttgatctgct gcttcgtgtg tttcagcaac gcggcctgct tggcctcgct
     6721 gacctgtttt gccaggtcct cgccggcggt ttttcgcttc ttggtcgtca tagttcctcg
     6781 cgtgtcgatg gtcatcgact tcgccaaacc tgccgcctcc tgttcaagac gacgcgaacg
     6841 ctccacggcg gccgatggcg cgggcagggc agggggagcc agttgcacgc tgtcgcgctc
     6901 gatcttggcc gtagcttgct ggaccatcga gccgacggac tggaaggttt cgcggggcgc
     6961 acgcatgacg gtgcggcttg cgatggtttc ggcatcctcg gcggaaaacc ccgcgtcgat
     7021 cagttcttgc ctgtatgcct tccggtcaaa cgtccgattc attcaccctc cttgcgggat
     7081 tgccccgact cacgccgggg caatgtgccc ttattcctga tttgacccgc ctggtgcctt
     7141 ggtgtccaga taatccacct tatcggcaat gaagtcggtc ccgtagaccg tctggccgtc
     7201 cttctcgtac ttggtattcc gaatcttgcc ctgcacgaat accagcgacc ccttgcccaa
     7261 atacttgccg tgggcctcgg cctgagagcc aaaacacttg atgcggaaga agtcggtgcg
     7321 ctcctgcttg tcgccggcat cgttgcgcca catctaggat ctgccaggaa ccgtaaaaag
     7381 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     7441 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     7501 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     7561 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     7621 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     7681 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     7741 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     7801 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     7861 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     7921 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     7981 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     8041 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     8101 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     8161 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     8221 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     8281 gctgcaatga taccgcgaga accacgctca ccggctccag atttatcagc aataaaccag
     8341 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     8401 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     8461 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     8521 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     8581 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     8641 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     8701 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     8761 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     8821 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     8881 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     8941 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     9001 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcaaggttat
     9061 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     9121 cgcacgaatt ggccagcgct gccatttttg gggtgaggcc gttcgcggcc gaggggcgca
     9181 gcccctgggg ggatgggagg cccgcgttag cgggccggga gggttcgaga agggggggca
     9241 ccccccttcg gcgtgcgcgg tcacgcgcac agggcgcagc cctggttaaa aacaaggttt
     9301 ataaatattg gtttaaaagc aggttaaaag acaggttagc ggtggccgaa aaacgggcgg
     9361 aaacccttgc aaatgctgga ttttctgcct gtggacagcc cctcaaatgt caataggtgc
     9421 gcccctcatc tgtcagcact ctgcccctca agtgtcaagg atcgcgcccc tcatctgtca
     9481 gtagtcgcgc ccctcaagtg tcaataccgc agggcactta tccccaggct tgtccacatc
     9541 atctgtggga aactcgcgta aaatcaggcg ttttcgccga tttgcgaggc tggccagctc
     9601 cacgtcgccg gccgaaatcg agcctgcccc tcatctgtca acgccgcgcc gggtgagtcg
     9661 gcccctcaag tgtcaacgtc cgcccctcat ctgtcagtga gggccaagtt ttccgcgagg
     9721 tatccacaac gccggcggcc gcggtgtctc gcacacggct tcgacggcgt ttctggcgcg
     9781 tttgcagggc catagacggc cgccagccca gcggcgaggg caaccagccc ggtgagcgtc
     9841 gcaaaggaga tcctgatctg actgatgggc tgcctgtatc gagtggtgat tttgtgccga
     9901 gctgccggtc ggggagctgt tggctggctg gtggcaggat atattgtggt gtaaacaaat
     9961 tgacgcttag acaacttaat aacacattgc ggacgttttt aatgtactgg ggtggatgca
    10021 gtgggcccca c
//
