LOCUS       sequence-228054-        4882 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level1 Golden Gate Cassette: extended stem sgRNA cassette with 
            AtU6-26 terminator for SpCas9, targeting NbPDS gene in N. 
            benthamiana.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4882)
  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 4882)
  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: pEPOR1CB0023
FEATURES             Location/Qualifiers
     source          1..4882
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    585..609
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(765..1913)
                     /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(2249..2837)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2329..2348)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3008..3799,3800..3868))
                     /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     3631..3650
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3869..3973)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4060..4691
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    4783..4807
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 tctgtgaaga caaactagaa ttcgagctcg gagggagcat cttcattctt aagatatgaa
       61 gataatcttc aaaaggcccc tgggaatctg aaagaagaga agcaggccca tttatatggg
      121 aaagaacaat agtatttctt atataggccc atttaagttg aaaacaatct tcaaaagtcc
      181 cacatcgctt agataagaaa acgaagctga gtttatatac agctagagtc gaagtagtga
      241 ttgccgttaa tttgagagtc cagtttaaga gctatgctgg aaacagcata gcaagtttaa
      301 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt tttttgcaaa
      361 attttccaga tcgatttctt cttcctctgt tcttcggcgt tcaatttctg gggttttctc
      421 ttcgttttct gtaactgaaa cctaaaattt gacctaaaaa aaatctcaaa taatatgatt
      481 cagtggtttt gtacttttca gttagttgag ttttgcagtt ccgatgagat aaaccaatac
      541 gctttacttg tcttctgcac gaagtggttt aaactatcag tgtttgacag gatatattgg
      601 cgggtaaacc taagagaaaa gagcgtttat tagaataatc ggatatttaa aagggcgtga
      661 aaaggtttat ccgttcgtcc atttgtatgt gcatgccaac cacagggttc cccagatcag
      721 gcgctggctg ctgaaccccc agccggaact gaccccacaa ggccctagcg tttgcaatgc
      781 accaggtcat cattgaccca ggcgtgttcc accaggccgc tgcctcgcaa ctcttcgcag
      841 gcttcgccga cctgctcgcg ccacttcttc acgcgggtgg aatccgatcc gcacatgagg
      901 cggaaggttt ccagcttgag cgggtacggc tcccggtgcg agctgaaata gtcgaacatc
      961 cgtcgggccg tcggcgacag cttgcggtac ttctcccata tgaatttcgt gtagtggtcg
     1021 ccagcaaaca gcacgacgat ttcctcgtcg atcaggacct ggcaacggga cgttttcttg
     1081 ccacggtcca ggacgcggaa gcggtgcagc agcgacaccg attccaggtg cccaacgcgg
     1141 tcggacgtga agcccatcgc cgtcgcctgt aggcgcgaca ggcattcctc ggccttcgtg
     1201 taataccggc cattgatcga ccagcccagg tcctggcaaa gctcgtagaa cgtgaaggtg
     1261 atcggctcgc cgataggggt gcgcttcgcg tactccaaca cctgctgcca caccagttcg
     1321 tcatcgtcgg cccgcagctc gacgccggtg taggtgatct tcacgtcctt gttgacgtgg
     1381 aaaatgacct tgttttgcag cgcctcgcgc gggattttct tgttgcgcgt ggtgaacagg
     1441 gcagagcggg ccgtgtcgtt tggcatcgct cgcatcgtgt ccggccacgg cgcaatatcg
     1501 aacaaggaaa gctgcatttc cttgatctgc tgcttcgtgt gtttcagcaa cgcggcctgc
     1561 ttggcctcgc tgacctgttt tgccaggtcc tcgccggcgg tttttcgctt cttggtcgtc
     1621 atagttcctc gcgtgtcgat ggtcatcgac ttcgccaaac ctgccgcctc ctgttcaaga
     1681 cgacgcgaac gctccacggc ggccgatggc gcgggcaggg cagggggagc cagttgcacg
     1741 ctgtcgcgct cgatcttggc cgtagcttgc tggaccatcg agccgacgga ctggaaggtt
     1801 tcgcggggcg cacgcatgac ggtgcggctt gcgatggttt cggcatcctc ggcggaaaac
     1861 cccgcgtcga tcagttcttg cctgtatgcc ttccggtcaa acgtccgatt cattcaccct
     1921 ccttgcggga ttgccccgac tcacgccggg gcaatgtgcc cttattcctg atttgacccg
     1981 cctggtgcct tggtgtccag ataatccacc ttatcggcaa tgaagtcggt cccgtagacc
     2041 gtctggccgt ccttctcgta cttggtattc cgaatcttgc cctgcacgaa taccagcgac
     2101 cccttgccca aatacttgcc gtgggcctcg gcctgagagc caaaacactt gatgcggaag
     2161 aagtcggtgc gctcctgctt gtcgccggca tcgttgcgcc acatctagga tctgccagga
     2221 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     2281 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     2341 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     2401 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     2461 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     2521 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     2581 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     2641 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     2701 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     2761 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     2821 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     2881 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     2941 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     3001 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     3061 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     3121 ctggccccag tgctgcaatg ataccgcgag aaccacgctc accggctcca gatttatcag
     3181 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     3241 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     3301 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     3361 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     3421 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     3481 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     3541 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     3601 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     3661 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     3721 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     3781 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     3841 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     3901 atcaaggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     3961 taggggttcc gcgcacgaat tggccagcgc tgccattttt ggggtgaggc cgttcgcggc
     4021 cgaggggcgc agcccctggg gggatgggag gcccgcgtta gcgggccggg agggttcgag
     4081 aagggggggc accccccttc ggcgtgcgcg gtcacgcgca cagggcgcag ccctggttaa
     4141 aaacaaggtt tataaatatt ggtttaaaag caggttaaaa gacaggttag cggtggccga
     4201 aaaacgggcg gaaacccttg caaatgctgg attttctgcc tgtggacagc ccctcaaatg
     4261 tcaataggtg cgcccctcat ctgtcagcac tctgcccctc aagtgtcaag gatcgcgccc
     4321 ctcatctgtc agtagtcgcg cccctcaagt gtcaataccg cagggcactt atccccaggc
     4381 ttgtccacat catctgtggg aaactcgcgt aaaatcaggc gttttcgccg atttgcgagg
     4441 ctggccagct ccacgtcgcc ggccgaaatc gagcctgccc ctcatctgtc aacgccgcgc
     4501 cgggtgagtc ggcccctcaa gtgtcaacgt ccgcccctca tctgtcagtg agggccaagt
     4561 tttccgcgag gtatccacaa cgccggcggc cgcggtgtct cgcacacggc ttcgacggcg
     4621 tttctggcgc gtttgcaggg ccatagacgg ccgccagccc agcggcgagg gcaaccagcc
     4681 cggtgagcgt cgcaaaggag atcctgatct gactgatggg ctgcctgtat cgagtggtga
     4741 ttttgtgccg agctgccggt cggggagctg ttggctggct ggtggcagga tatattgtgg
     4801 tgtaaacaaa ttgacgctta gacaacttaa taacacattg cggacgtttt taatgtactg
     4861 gggtggatgc agtgggcccc ac
//
