LOCUS       sequence-228057-        4693 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level1 Golden Gate Cassette: sgRNA cassette for SaCas9, targeting 
            NbPDS gene in N. benthamiana.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4693)
  AUTHORS   Raitskin O, Schudoma C, West A, Patron NJ
  TITLE     Comparison of efficiency and specificity of CRISPR-associated (Cas) 
            nucleases in plants: An expanded toolkit for precision genome 
            engineering.
  JOURNAL   PLoS One. 2019 Feb 27;14(2):e0211598. doi: 
            10.1371/journal.pone.0211598. eCollection 2019.
  PUBMED    30811422
REFERENCE   2  (bases 1 to 4693)
  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: "PLoS One.";
            date: "2019-02-27"; volume: "14(2):e0211598. doi"; pages: " 
            10.1371/journal.pone.0211598. eCollection 2019"
COMMENT     Sequence Label: pEPOR1CB0058
FEATURES             Location/Qualifiers
     source          1..4693
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        260..335
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     misc_feature    396..420
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(576..1724)
                     /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(2060..2648)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2140..2159)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2819..3610,3611..3679))
                     /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     3442..3461
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3680..3784)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3871..4502
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    4594..4618
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 tctgtgaaga caaactagaa ttcgagctcg gagcatcttc attcttaaga tatgaagata
       61 atcttcaaaa ggcccctggg aatctgaaag aagagaagca ggcccattta tatgggaaag
      121 aacaatagta tttcttatat aggcccattt aagttgaaaa caatcttcaa aagtcccaca
      181 tcgcttagat aagaaaacga agctgagttt atatacagct agagtcgaag tagtgattgt
      241 atcttcggcc atgaagctgg ttttagtact ctggaaacag aatctactaa aacaaggcaa
      301 aatgccgtgt ttatctcgtc aacttgttgg cgagattttt ttacttgtct cgctttactt
      361 gtcttctgca cgaagtggtt taaactatca gtgtttgaca ggatatattg gcgggtaaac
      421 ctaagagaaa agagcgttta ttagaataat cggatattta aaagggcgtg aaaaggttta
      481 tccgttcgtc catttgtatg tgcatgccaa ccacagggtt ccccagatca ggcgctggct
      541 gctgaacccc cagccggaac tgaccccaca aggccctagc gtttgcaatg caccaggtca
      601 tcattgaccc aggcgtgttc caccaggccg ctgcctcgca actcttcgca ggcttcgccg
      661 acctgctcgc gccacttctt cacgcgggtg gaatccgatc cgcacatgag gcggaaggtt
      721 tccagcttga gcgggtacgg ctcccggtgc gagctgaaat agtcgaacat ccgtcgggcc
      781 gtcggcgaca gcttgcggta cttctcccat atgaatttcg tgtagtggtc gccagcaaac
      841 agcacgacga tttcctcgtc gatcaggacc tggcaacggg acgttttctt gccacggtcc
      901 aggacgcgga agcggtgcag cagcgacacc gattccaggt gcccaacgcg gtcggacgtg
      961 aagcccatcg ccgtcgcctg taggcgcgac aggcattcct cggccttcgt gtaataccgg
     1021 ccattgatcg accagcccag gtcctggcaa agctcgtaga acgtgaaggt gatcggctcg
     1081 ccgatagggg tgcgcttcgc gtactccaac acctgctgcc acaccagttc gtcatcgtcg
     1141 gcccgcagct cgacgccggt gtaggtgatc ttcacgtcct tgttgacgtg gaaaatgacc
     1201 ttgttttgca gcgcctcgcg cgggattttc ttgttgcgcg tggtgaacag ggcagagcgg
     1261 gccgtgtcgt ttggcatcgc tcgcatcgtg tccggccacg gcgcaatatc gaacaaggaa
     1321 agctgcattt ccttgatctg ctgcttcgtg tgtttcagca acgcggcctg cttggcctcg
     1381 ctgacctgtt ttgccaggtc ctcgccggcg gtttttcgct tcttggtcgt catagttcct
     1441 cgcgtgtcga tggtcatcga cttcgccaaa cctgccgcct cctgttcaag acgacgcgaa
     1501 cgctccacgg cggccgatgg cgcgggcagg gcagggggag ccagttgcac gctgtcgcgc
     1561 tcgatcttgg ccgtagcttg ctggaccatc gagccgacgg actggaaggt ttcgcggggc
     1621 gcacgcatga cggtgcggct tgcgatggtt tcggcatcct cggcggaaaa ccccgcgtcg
     1681 atcagttctt gcctgtatgc cttccggtca aacgtccgat tcattcaccc tccttgcggg
     1741 attgccccga ctcacgccgg ggcaatgtgc ccttattcct gatttgaccc gcctggtgcc
     1801 ttggtgtcca gataatccac cttatcggca atgaagtcgg tcccgtagac cgtctggccg
     1861 tccttctcgt acttggtatt ccgaatcttg ccctgcacga ataccagcga ccccttgccc
     1921 aaatacttgc cgtgggcctc ggcctgagag ccaaaacact tgatgcggaa gaagtcggtg
     1981 cgctcctgct tgtcgccggc atcgttgcgc cacatctagg atctgccagg aaccgtaaaa
     2041 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2101 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2161 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     2221 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     2281 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     2341 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     2401 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     2461 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     2521 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     2581 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     2641 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     2701 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     2761 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     2821 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     2881 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     2941 gtgctgcaat gataccgcga gaaccacgct caccggctcc agatttatca gcaataaacc
     3001 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     3061 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     3121 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     3181 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     3241 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     3301 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     3361 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     3421 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     3481 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     3541 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     3601 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     3661 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcaaggtt
     3721 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     3781 cgcgcacgaa ttggccagcg ctgccatttt tggggtgagg ccgttcgcgg ccgaggggcg
     3841 cagcccctgg ggggatggga ggcccgcgtt agcgggccgg gagggttcga gaaggggggg
     3901 cacccccctt cggcgtgcgc ggtcacgcgc acagggcgca gccctggtta aaaacaaggt
     3961 ttataaatat tggtttaaaa gcaggttaaa agacaggtta gcggtggccg aaaaacgggc
     4021 ggaaaccctt gcaaatgctg gattttctgc ctgtggacag cccctcaaat gtcaataggt
     4081 gcgcccctca tctgtcagca ctctgcccct caagtgtcaa ggatcgcgcc cctcatctgt
     4141 cagtagtcgc gcccctcaag tgtcaatacc gcagggcact tatccccagg cttgtccaca
     4201 tcatctgtgg gaaactcgcg taaaatcagg cgttttcgcc gatttgcgag gctggccagc
     4261 tccacgtcgc cggccgaaat cgagcctgcc cctcatctgt caacgccgcg ccgggtgagt
     4321 cggcccctca agtgtcaacg tccgcccctc atctgtcagt gagggccaag ttttccgcga
     4381 ggtatccaca acgccggcgg ccgcggtgtc tcgcacacgg cttcgacggc gtttctggcg
     4441 cgtttgcagg gccatagacg gccgccagcc cagcggcgag ggcaaccagc ccggtgagcg
     4501 tcgcaaagga gatcctgatc tgactgatgg gctgcctgta tcgagtggtg attttgtgcc
     4561 gagctgccgg tcggggagct gttggctggc tggtggcagg atatattgtg gtgtaaacaa
     4621 attgacgctt agacaactta ataacacatt gcggacgttt ttaatgtact ggggtggatg
     4681 cagtgggccc cac
//
