LOCUS       sequence-228059-        4632 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level1 Golden Gate Cassette: sgRNA cassette for AsCas12a, targeting 
            NbPDS gene in N. benthamiana.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4632)
  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 4632)
  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: pEPOR1CB0028
FEATURES             Location/Qualifiers
     source          1..4632
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    335..359
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(515..1663)
                     /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(1999..2587)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2079..2098)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2758..3549,3550..3618))
                     /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     3381..3400
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3619..3723)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3810..4441
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    4533..4557
                     /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 aatttctact cttgtagata cgtatcttcg gccatgaagc tgtttttttc gctttacttg
      301 tcttctgcac gaagtggttt aaactatcag tgtttgacag gatatattgg cgggtaaacc
      361 taagagaaaa gagcgtttat tagaataatc ggatatttaa aagggcgtga aaaggtttat
      421 ccgttcgtcc atttgtatgt gcatgccaac cacagggttc cccagatcag gcgctggctg
      481 ctgaaccccc agccggaact gaccccacaa ggccctagcg tttgcaatgc accaggtcat
      541 cattgaccca ggcgtgttcc accaggccgc tgcctcgcaa ctcttcgcag gcttcgccga
      601 cctgctcgcg ccacttcttc acgcgggtgg aatccgatcc gcacatgagg cggaaggttt
      661 ccagcttgag cgggtacggc tcccggtgcg agctgaaata gtcgaacatc cgtcgggccg
      721 tcggcgacag cttgcggtac ttctcccata tgaatttcgt gtagtggtcg ccagcaaaca
      781 gcacgacgat ttcctcgtcg atcaggacct ggcaacggga cgttttcttg ccacggtcca
      841 ggacgcggaa gcggtgcagc agcgacaccg attccaggtg cccaacgcgg tcggacgtga
      901 agcccatcgc cgtcgcctgt aggcgcgaca ggcattcctc ggccttcgtg taataccggc
      961 cattgatcga ccagcccagg tcctggcaaa gctcgtagaa cgtgaaggtg atcggctcgc
     1021 cgataggggt gcgcttcgcg tactccaaca cctgctgcca caccagttcg tcatcgtcgg
     1081 cccgcagctc gacgccggtg taggtgatct tcacgtcctt gttgacgtgg aaaatgacct
     1141 tgttttgcag cgcctcgcgc gggattttct tgttgcgcgt ggtgaacagg gcagagcggg
     1201 ccgtgtcgtt tggcatcgct cgcatcgtgt ccggccacgg cgcaatatcg aacaaggaaa
     1261 gctgcatttc cttgatctgc tgcttcgtgt gtttcagcaa cgcggcctgc ttggcctcgc
     1321 tgacctgttt tgccaggtcc tcgccggcgg tttttcgctt cttggtcgtc atagttcctc
     1381 gcgtgtcgat ggtcatcgac ttcgccaaac ctgccgcctc ctgttcaaga cgacgcgaac
     1441 gctccacggc ggccgatggc gcgggcaggg cagggggagc cagttgcacg ctgtcgcgct
     1501 cgatcttggc cgtagcttgc tggaccatcg agccgacgga ctggaaggtt tcgcggggcg
     1561 cacgcatgac ggtgcggctt gcgatggttt cggcatcctc ggcggaaaac cccgcgtcga
     1621 tcagttcttg cctgtatgcc ttccggtcaa acgtccgatt cattcaccct ccttgcggga
     1681 ttgccccgac tcacgccggg gcaatgtgcc cttattcctg atttgacccg cctggtgcct
     1741 tggtgtccag ataatccacc ttatcggcaa tgaagtcggt cccgtagacc gtctggccgt
     1801 ccttctcgta cttggtattc cgaatcttgc cctgcacgaa taccagcgac cccttgccca
     1861 aatacttgcc gtgggcctcg gcctgagagc caaaacactt gatgcggaag aagtcggtgc
     1921 gctcctgctt gtcgccggca tcgttgcgcc acatctagga tctgccagga accgtaaaaa
     1981 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     2041 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     2101 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     2161 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     2221 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     2281 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     2341 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     2401 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     2461 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     2521 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     2581 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     2641 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     2701 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     2761 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     2821 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     2881 tgctgcaatg ataccgcgag aaccacgctc accggctcca gatttatcag caataaacca
     2941 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     3001 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     3061 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     3121 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     3181 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     3241 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     3301 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     3361 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     3421 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     3481 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     3541 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     3601 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcaaggtta
     3661 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     3721 gcgcacgaat tggccagcgc tgccattttt ggggtgaggc cgttcgcggc cgaggggcgc
     3781 agcccctggg gggatgggag gcccgcgtta gcgggccggg agggttcgag aagggggggc
     3841 accccccttc ggcgtgcgcg gtcacgcgca cagggcgcag ccctggttaa aaacaaggtt
     3901 tataaatatt ggtttaaaag caggttaaaa gacaggttag cggtggccga aaaacgggcg
     3961 gaaacccttg caaatgctgg attttctgcc tgtggacagc ccctcaaatg tcaataggtg
     4021 cgcccctcat ctgtcagcac tctgcccctc aagtgtcaag gatcgcgccc ctcatctgtc
     4081 agtagtcgcg cccctcaagt gtcaataccg cagggcactt atccccaggc ttgtccacat
     4141 catctgtggg aaactcgcgt aaaatcaggc gttttcgccg atttgcgagg ctggccagct
     4201 ccacgtcgcc ggccgaaatc gagcctgccc ctcatctgtc aacgccgcgc cgggtgagtc
     4261 ggcccctcaa gtgtcaacgt ccgcccctca tctgtcagtg agggccaagt tttccgcgag
     4321 gtatccacaa cgccggcggc cgcggtgtct cgcacacggc ttcgacggcg tttctggcgc
     4381 gtttgcaggg ccatagacgg ccgccagccc agcggcgagg gcaaccagcc cggtgagcgt
     4441 cgcaaaggag atcctgatct gactgatggg ctgcctgtat cgagtggtga ttttgtgccg
     4501 agctgccggt cggggagctg ttggctggct ggtggcagga tatattgtgg tgtaaacaaa
     4561 ttgacgctta gacaacttaa taacacattg cggacgtttt taatgtactg gggtggatgc
     4621 agtgggcccc ac
//
