LOCUS       sequence-221822-        4718 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level1 Golden Gate Cassette: sgRNA cassette for spCas9, targeting 
            NbPDS gene in N. benthamiana with 1 mismatch at spacer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4718)
  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 4718)
  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: pEPOR1CB0020
FEATURES             Location/Qualifiers
     source          1..4718
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        259..334
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(346..370)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    421..445
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(601..1749)
                     /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(2085..2673)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2165..2184)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2844..3635,3636..3704))
                     /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     3467..3486
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3705..3809)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3896..4527
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    4619..4643
                     /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 tagtgattgc
      241 cgttaatttg ggagtccagt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
      301 ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tctagaccca gctttcttgt
      361 acaaagttgg cattacgctt tacttgtctt ctgcacgaag tggtttaaac tatcagtgtt
      421 tgacaggata tattggcggg taaacctaag agaaaagagc gtttattaga ataatcggat
      481 atttaaaagg gcgtgaaaag gtttatccgt tcgtccattt gtatgtgcat gccaaccaca
      541 gggttcccca gatcaggcgc tggctgctga acccccagcc ggaactgacc ccacaaggcc
      601 ctagcgtttg caatgcacca ggtcatcatt gacccaggcg tgttccacca ggccgctgcc
      661 tcgcaactct tcgcaggctt cgccgacctg ctcgcgccac ttcttcacgc gggtggaatc
      721 cgatccgcac atgaggcgga aggtttccag cttgagcggg tacggctccc ggtgcgagct
      781 gaaatagtcg aacatccgtc gggccgtcgg cgacagcttg cggtacttct cccatatgaa
      841 tttcgtgtag tggtcgccag caaacagcac gacgatttcc tcgtcgatca ggacctggca
      901 acgggacgtt ttcttgccac ggtccaggac gcggaagcgg tgcagcagcg acaccgattc
      961 caggtgccca acgcggtcgg acgtgaagcc catcgccgtc gcctgtaggc gcgacaggca
     1021 ttcctcggcc ttcgtgtaat accggccatt gatcgaccag cccaggtcct ggcaaagctc
     1081 gtagaacgtg aaggtgatcg gctcgccgat aggggtgcgc ttcgcgtact ccaacacctg
     1141 ctgccacacc agttcgtcat cgtcggcccg cagctcgacg ccggtgtagg tgatcttcac
     1201 gtccttgttg acgtggaaaa tgaccttgtt ttgcagcgcc tcgcgcggga ttttcttgtt
     1261 gcgcgtggtg aacagggcag agcgggccgt gtcgtttggc atcgctcgca tcgtgtccgg
     1321 ccacggcgca atatcgaaca aggaaagctg catttccttg atctgctgct tcgtgtgttt
     1381 cagcaacgcg gcctgcttgg cctcgctgac ctgttttgcc aggtcctcgc cggcggtttt
     1441 tcgcttcttg gtcgtcatag ttcctcgcgt gtcgatggtc atcgacttcg ccaaacctgc
     1501 cgcctcctgt tcaagacgac gcgaacgctc cacggcggcc gatggcgcgg gcagggcagg
     1561 gggagccagt tgcacgctgt cgcgctcgat cttggccgta gcttgctgga ccatcgagcc
     1621 gacggactgg aaggtttcgc ggggcgcacg catgacggtg cggcttgcga tggtttcggc
     1681 atcctcggcg gaaaaccccg cgtcgatcag ttcttgcctg tatgccttcc ggtcaaacgt
     1741 ccgattcatt caccctcctt gcgggattgc cccgactcac gccggggcaa tgtgccctta
     1801 ttcctgattt gacccgcctg gtgccttggt gtccagataa tccaccttat cggcaatgaa
     1861 gtcggtcccg tagaccgtct ggccgtcctt ctcgtacttg gtattccgaa tcttgccctg
     1921 cacgaatacc agcgacccct tgcccaaata cttgccgtgg gcctcggcct gagagccaaa
     1981 acacttgatg cggaagaagt cggtgcgctc ctgcttgtcg ccggcatcgt tgcgccacat
     2041 ctaggatctg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     2101 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     2161 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     2221 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     2281 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     2341 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     2401 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     2461 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     2521 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     2581 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     2641 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     2701 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     2761 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     2821 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     2881 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     2941 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaacc acgctcaccg
     3001 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     3061 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     3121 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     3181 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     3241 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     3301 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     3361 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     3421 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     3481 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     3541 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     3601 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     3661 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     3721 tattattgaa gcatttatca aggttattgt ctcatgagcg gatacatatt tgaatgtatt
     3781 tagaaaaata aacaaatagg ggttccgcgc acgaattggc cagcgctgcc atttttgggg
     3841 tgaggccgtt cgcggccgag gggcgcagcc cctgggggga tgggaggccc gcgttagcgg
     3901 gccgggaggg ttcgagaagg gggggcaccc cccttcggcg tgcgcggtca cgcgcacagg
     3961 gcgcagccct ggttaaaaac aaggtttata aatattggtt taaaagcagg ttaaaagaca
     4021 ggttagcggt ggccgaaaaa cgggcggaaa cccttgcaaa tgctggattt tctgcctgtg
     4081 gacagcccct caaatgtcaa taggtgcgcc cctcatctgt cagcactctg cccctcaagt
     4141 gtcaaggatc gcgcccctca tctgtcagta gtcgcgcccc tcaagtgtca ataccgcagg
     4201 gcacttatcc ccaggcttgt ccacatcatc tgtgggaaac tcgcgtaaaa tcaggcgttt
     4261 tcgccgattt gcgaggctgg ccagctccac gtcgccggcc gaaatcgagc ctgcccctca
     4321 tctgtcaacg ccgcgccggg tgagtcggcc cctcaagtgt caacgtccgc ccctcatctg
     4381 tcagtgaggg ccaagttttc cgcgaggtat ccacaacgcc ggcggccgcg gtgtctcgca
     4441 cacggcttcg acggcgtttc tggcgcgttt gcagggccat agacggccgc cagcccagcg
     4501 gcgagggcaa ccagcccggt gagcgtcgca aaggagatcc tgatctgact gatgggctgc
     4561 ctgtatcgag tggtgatttt gtgccgagct gccggtcggg gagctgttgg ctggctggtg
     4621 gcaggatata ttgtggtgta aacaaattga cgcttagaca acttaataac acattgcgga
     4681 cgtttttaat gtactggggt ggatgcagtg ggccccac
//
