LOCUS       sequence-221838-        4694 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level1 Golden Gate Cassette: sgRNA cassette for spCas9, targeting 
            Protein kinase superfamily protein {AT1G73460} in A. thaliana.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4694)
  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 4694)
  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: pEPOR1CB0099
FEATURES             Location/Qualifiers
     source          1..4694
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        261..336
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     misc_feature    397..421
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(577..1725)
                     /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(2061..2649)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2141..2160)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2820..3611,3612..3680))
                     /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     3443..3462
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3681..3785)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3872..4503
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    4595..4619
                     /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 tcatggtgat caggacttcc gttttagtac tctggaaaca gaatctacta aaacaaggca
      301 aaatgccgtg tttatctcgt caacttgttg gcgagatttt tttacttgtc tcgctttact
      361 tgtcttctgc acgaagtggt ttaaactatc agtgtttgac aggatatatt ggcgggtaaa
      421 cctaagagaa aagagcgttt attagaataa tcggatattt aaaagggcgt gaaaaggttt
      481 atccgttcgt ccatttgtat gtgcatgcca accacagggt tccccagatc aggcgctggc
      541 tgctgaaccc ccagccggaa ctgaccccac aaggccctag cgtttgcaat gcaccaggtc
      601 atcattgacc caggcgtgtt ccaccaggcc gctgcctcgc aactcttcgc aggcttcgcc
      661 gacctgctcg cgccacttct tcacgcgggt ggaatccgat ccgcacatga ggcggaaggt
      721 ttccagcttg agcgggtacg gctcccggtg cgagctgaaa tagtcgaaca tccgtcgggc
      781 cgtcggcgac agcttgcggt acttctccca tatgaatttc gtgtagtggt cgccagcaaa
      841 cagcacgacg atttcctcgt cgatcaggac ctggcaacgg gacgttttct tgccacggtc
      901 caggacgcgg aagcggtgca gcagcgacac cgattccagg tgcccaacgc ggtcggacgt
      961 gaagcccatc gccgtcgcct gtaggcgcga caggcattcc tcggccttcg tgtaataccg
     1021 gccattgatc gaccagccca ggtcctggca aagctcgtag aacgtgaagg tgatcggctc
     1081 gccgataggg gtgcgcttcg cgtactccaa cacctgctgc cacaccagtt cgtcatcgtc
     1141 ggcccgcagc tcgacgccgg tgtaggtgat cttcacgtcc ttgttgacgt ggaaaatgac
     1201 cttgttttgc agcgcctcgc gcgggatttt cttgttgcgc gtggtgaaca gggcagagcg
     1261 ggccgtgtcg tttggcatcg ctcgcatcgt gtccggccac ggcgcaatat cgaacaagga
     1321 aagctgcatt tccttgatct gctgcttcgt gtgtttcagc aacgcggcct gcttggcctc
     1381 gctgacctgt tttgccaggt cctcgccggc ggtttttcgc ttcttggtcg tcatagttcc
     1441 tcgcgtgtcg atggtcatcg acttcgccaa acctgccgcc tcctgttcaa gacgacgcga
     1501 acgctccacg gcggccgatg gcgcgggcag ggcaggggga gccagttgca cgctgtcgcg
     1561 ctcgatcttg gccgtagctt gctggaccat cgagccgacg gactggaagg tttcgcgggg
     1621 cgcacgcatg acggtgcggc ttgcgatggt ttcggcatcc tcggcggaaa accccgcgtc
     1681 gatcagttct tgcctgtatg ccttccggtc aaacgtccga ttcattcacc ctccttgcgg
     1741 gattgccccg actcacgccg gggcaatgtg cccttattcc tgatttgacc cgcctggtgc
     1801 cttggtgtcc agataatcca ccttatcggc aatgaagtcg gtcccgtaga ccgtctggcc
     1861 gtccttctcg tacttggtat tccgaatctt gccctgcacg aataccagcg accccttgcc
     1921 caaatacttg ccgtgggcct cggcctgaga gccaaaacac ttgatgcgga agaagtcggt
     1981 gcgctcctgc ttgtcgccgg catcgttgcg ccacatctag gatctgccag gaaccgtaaa
     2041 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     2101 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     2161 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     2221 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     2281 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     2341 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     2401 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     2461 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     2521 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     2581 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     2641 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     2701 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     2761 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     2821 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     2881 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     2941 agtgctgcaa tgataccgcg agaaccacgc tcaccggctc cagatttatc agcaataaac
     3001 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     3061 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     3121 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     3181 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     3241 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     3301 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     3361 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     3421 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     3481 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     3541 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     3601 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     3661 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcaaggt
     3721 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     3781 ccgcgcacga attggccagc gctgccattt ttggggtgag gccgttcgcg gccgaggggc
     3841 gcagcccctg gggggatggg aggcccgcgt tagcgggccg ggagggttcg agaagggggg
     3901 gcacccccct tcggcgtgcg cggtcacgcg cacagggcgc agccctggtt aaaaacaagg
     3961 tttataaata ttggtttaaa agcaggttaa aagacaggtt agcggtggcc gaaaaacggg
     4021 cggaaaccct tgcaaatgct ggattttctg cctgtggaca gcccctcaaa tgtcaatagg
     4081 tgcgcccctc atctgtcagc actctgcccc tcaagtgtca aggatcgcgc ccctcatctg
     4141 tcagtagtcg cgcccctcaa gtgtcaatac cgcagggcac ttatccccag gcttgtccac
     4201 atcatctgtg ggaaactcgc gtaaaatcag gcgttttcgc cgatttgcga ggctggccag
     4261 ctccacgtcg ccggccgaaa tcgagcctgc ccctcatctg tcaacgccgc gccgggtgag
     4321 tcggcccctc aagtgtcaac gtccgcccct catctgtcag tgagggccaa gttttccgcg
     4381 aggtatccac aacgccggcg gccgcggtgt ctcgcacacg gcttcgacgg cgtttctggc
     4441 gcgtttgcag ggccatagac ggccgccagc ccagcggcga gggcaaccag cccggtgagc
     4501 gtcgcaaagg agatcctgat ctgactgatg ggctgcctgt atcgagtggt gattttgtgc
     4561 cgagctgccg gtcggggagc tgttggctgg ctggtggcag gatatattgt ggtgtaaaca
     4621 aattgacgct tagacaactt aataacacat tgcggacgtt tttaatgtac tggggtggat
     4681 gcagtgggcc ccac
//
