LOCUS       sequence-228165-        4719 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level1 Golden Gate Cassette: sgRNA cassette for spCas9, targeting 
            {AT1G68250} in A. thaliana.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4719)
  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 4719)
  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: pEPOR1CB0089
FEATURES             Location/Qualifiers
     source          1..4719
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        260..335
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(347..371)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    422..446
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(602..1750)
                     /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(2086..2674)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2166..2185)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2845..3636,3637..3705))
                     /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     3468..3487
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3706..3810)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3897..4528
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    4620..4644
                     /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 gagccggctg agccatggag ttttagagct agaaatagca agttaaaata aggctagtcc
      301 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttctagaccc agctttcttg
      361 tacaaagttg gcattacgct ttacttgtct tctgcacgaa gtggtttaaa ctatcagtgt
      421 ttgacaggat atattggcgg gtaaacctaa gagaaaagag cgtttattag aataatcgga
      481 tatttaaaag ggcgtgaaaa ggtttatccg ttcgtccatt tgtatgtgca tgccaaccac
      541 agggttcccc agatcaggcg ctggctgctg aacccccagc cggaactgac cccacaaggc
      601 cctagcgttt gcaatgcacc aggtcatcat tgacccaggc gtgttccacc aggccgctgc
      661 ctcgcaactc ttcgcaggct tcgccgacct gctcgcgcca cttcttcacg cgggtggaat
      721 ccgatccgca catgaggcgg aaggtttcca gcttgagcgg gtacggctcc cggtgcgagc
      781 tgaaatagtc gaacatccgt cgggccgtcg gcgacagctt gcggtacttc tcccatatga
      841 atttcgtgta gtggtcgcca gcaaacagca cgacgatttc ctcgtcgatc aggacctggc
      901 aacgggacgt tttcttgcca cggtccagga cgcggaagcg gtgcagcagc gacaccgatt
      961 ccaggtgccc aacgcggtcg gacgtgaagc ccatcgccgt cgcctgtagg cgcgacaggc
     1021 attcctcggc cttcgtgtaa taccggccat tgatcgacca gcccaggtcc tggcaaagct
     1081 cgtagaacgt gaaggtgatc ggctcgccga taggggtgcg cttcgcgtac tccaacacct
     1141 gctgccacac cagttcgtca tcgtcggccc gcagctcgac gccggtgtag gtgatcttca
     1201 cgtccttgtt gacgtggaaa atgaccttgt tttgcagcgc ctcgcgcggg attttcttgt
     1261 tgcgcgtggt gaacagggca gagcgggccg tgtcgtttgg catcgctcgc atcgtgtccg
     1321 gccacggcgc aatatcgaac aaggaaagct gcatttcctt gatctgctgc ttcgtgtgtt
     1381 tcagcaacgc ggcctgcttg gcctcgctga cctgttttgc caggtcctcg ccggcggttt
     1441 ttcgcttctt ggtcgtcata gttcctcgcg tgtcgatggt catcgacttc gccaaacctg
     1501 ccgcctcctg ttcaagacga cgcgaacgct ccacggcggc cgatggcgcg ggcagggcag
     1561 ggggagccag ttgcacgctg tcgcgctcga tcttggccgt agcttgctgg accatcgagc
     1621 cgacggactg gaaggtttcg cggggcgcac gcatgacggt gcggcttgcg atggtttcgg
     1681 catcctcggc ggaaaacccc gcgtcgatca gttcttgcct gtatgccttc cggtcaaacg
     1741 tccgattcat tcaccctcct tgcgggattg ccccgactca cgccggggca atgtgccctt
     1801 attcctgatt tgacccgcct ggtgccttgg tgtccagata atccacctta tcggcaatga
     1861 agtcggtccc gtagaccgtc tggccgtcct tctcgtactt ggtattccga atcttgccct
     1921 gcacgaatac cagcgacccc ttgcccaaat acttgccgtg ggcctcggcc tgagagccaa
     1981 aacacttgat gcggaagaag tcggtgcgct cctgcttgtc gccggcatcg ttgcgccaca
     2041 tctaggatct gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     2101 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     2161 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     2221 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     2281 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     2341 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     2401 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     2461 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     2521 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     2581 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     2641 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     2701 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     2761 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     2821 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     2881 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     2941 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagaac cacgctcacc
     3001 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     3061 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     3121 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     3181 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     3241 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     3301 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     3361 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     3421 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     3481 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     3541 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     3601 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     3661 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     3721 atattattga agcatttatc aaggttattg tctcatgagc ggatacatat ttgaatgtat
     3781 ttagaaaaat aaacaaatag gggttccgcg cacgaattgg ccagcgctgc catttttggg
     3841 gtgaggccgt tcgcggccga ggggcgcagc ccctgggggg atgggaggcc cgcgttagcg
     3901 ggccgggagg gttcgagaag ggggggcacc ccccttcggc gtgcgcggtc acgcgcacag
     3961 ggcgcagccc tggttaaaaa caaggtttat aaatattggt ttaaaagcag gttaaaagac
     4021 aggttagcgg tggccgaaaa acgggcggaa acccttgcaa atgctggatt ttctgcctgt
     4081 ggacagcccc tcaaatgtca ataggtgcgc ccctcatctg tcagcactct gcccctcaag
     4141 tgtcaaggat cgcgcccctc atctgtcagt agtcgcgccc ctcaagtgtc aataccgcag
     4201 ggcacttatc cccaggcttg tccacatcat ctgtgggaaa ctcgcgtaaa atcaggcgtt
     4261 ttcgccgatt tgcgaggctg gccagctcca cgtcgccggc cgaaatcgag cctgcccctc
     4321 atctgtcaac gccgcgccgg gtgagtcggc ccctcaagtg tcaacgtccg cccctcatct
     4381 gtcagtgagg gccaagtttt ccgcgaggta tccacaacgc cggcggccgc ggtgtctcgc
     4441 acacggcttc gacggcgttt ctggcgcgtt tgcagggcca tagacggccg ccagcccagc
     4501 ggcgagggca accagcccgg tgagcgtcgc aaaggagatc ctgatctgac tgatgggctg
     4561 cctgtatcga gtggtgattt tgtgccgagc tgccggtcgg ggagctgttg gctggctggt
     4621 ggcaggatat attgtggtgt aaacaaattg acgcttagac aacttaataa cacattgcgg
     4681 acgtttttaa tgtactgggg tggatgcagt gggccccac
//
