LOCUS       sequence-325662-        4595 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intermediate vector for eight gRNAs using GoldenGate, contains ccdB 
            and attL1-L2 sites for Gateway.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4595)
  AUTHORS   Ursache R, Fujita S, Denervaud Tendon V, Geldner N
  TITLE     Combined fluorescent seed selection and multiplex CRISPR/Cas9 
            assembly for fast generation of multiple Arabidopsis mutants.
  JOURNAL   Plant Methods. 2021 Oct 30;17(1):111. doi: 
            10.1186/s13007-021-00811-9.
  PUBMED    34717688
REFERENCE   2  (bases 1 to 4595)
  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: "Plant 
            Methods."; date: "2021-10-30"; volume: "17(1):111. doi"; pages: " 
            10.1186/s13007-021-00811-9"
COMMENT     Sequence Label: pRU325
FEATURES             Location/Qualifiers
     source          1..4595
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(1133..1438)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(1235..1254)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1783..2442)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2357..2376
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2443..2545)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(2720..2819)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2836..2855)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2837..2855)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2860..2876)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2860..2876)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2978..2997)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2989..3798
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3056..3075)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3945..4533
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4434..4453
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacacattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctta tacaaaagtt gccccatggc gttccctcta gataacgcag gatccccaag
      721 tggtggctat cgagaccgtc gaccgacagc cttccaaatg ttcttcgggt gatgctgcca
      781 acttagtcga ccgacagcct tccaaatgtt cttctcaaac ggaatcgtcg tatccagcct
      841 actcgctatt gtcctcaatg ccgtattaaa tcataaaaag aaataagaaa aagaggtgcg
      901 agcctctttt ttgtgtgaca aaataaaaac atctacctat tcatatacgc tagtgtcata
      961 gtcctgaaaa tcatctgcat caagaacaat ttcacaactc ttatactttt ctcttacaag
     1021 tcgttcggct tcatctggat tttcagcctc tatacttact aaacgtgata aagtttctgt
     1081 aatttctact gtatcgacct gcagactggc tgtgtataag ggagcctgac atttatattc
     1141 cccagaacat caggttaatg gcgtttttga tgtcattttc gcggtggctg agatcagcca
     1201 cttcttcccc gataacggag accggcacac tggccatatc ggtggtcatc atgcgccagc
     1261 tttcatcccc gatatgcacc accgggtaaa gttcacggga gactttatct gacagcagac
     1321 gtgcactggc cagggggatc accatccgtc gcccgggcgt gtcaataata tcactctgta
     1381 catccacaaa cagacgataa cggctctctc ttttataggt gtaaacctta aactgcattt
     1441 caccagcccc tgttctcgtc agcaaaagag ccgttcattt caataaaccg ggcgacctca
     1501 gccatccctt cctgattttc cgctttccag cgttcggcac gcagacgacg ggcttcattc
     1561 tgcatggttg tgcttaccag accggagata ttgacatcat atatgccttg agcaactgat
     1621 agctgtcgct gtcaactgtc actgtaatac gctgcttcat agcatacctc tttttgacat
     1681 acttcgggta tacatatcag tatatattct tataccgcaa aaatcagcgc gcaaatacgc
     1741 atactgttat ctggctttta gtaagccgga tccacgcggc gtttacgccc cgccctgcca
     1801 ctcatcgcag tactgttgta attcattaag cattctgccg acatggaagc catcacagac
     1861 ggcatgatga acctgaatcg ccagcggcat cagcaccttg tcgccttgcg tataatattt
     1921 gcccatggtg aaaacggggg cgaagaagtt gtccatattg gccacgttta aatcaaaact
     1981 ggtgaaactc acccagggat tggctgagac gaaaaacata ttctcaataa accctttagg
     2041 gaaataggcc aggttttcac cgtaacacgc cacatcttgc gaatatatgt gtagaaactg
     2101 ccggaaatcg tcgtggtatt cactccagag cgatgaaaac gtttcagttt gctcatggaa
     2161 aacggtgtaa caagggtgaa cactatccca tatcaccagc tcaccgtctt tcattgccat
     2221 acggaattcc ggatgagcat tcatcaggcg ggcaagaatg tgaataaagg ccggataaaa
     2281 cttgtgctta tttttcttta cggtctttaa aaaggccgta atatccagct gaacggtctg
     2341 gttataggta cattgagcaa ctgactgaaa tgcctcaaaa tgttctttac gatgccattg
     2401 ggatatatca acggtggtat atccagtgat ttttttctcc attttagctt ccttagctcc
     2461 tgaaaatctc gataactcaa aaaatacgcc cggtagtgat cttatttcat tatggtgaaa
     2521 gttggaacct cttacgtgcc gatcaacgtc tcattttcgc caaaagttgg cccagggctt
     2581 cccggtatca acagggacac caggatttat ttattctgcg aagtgatctt ccgtcacagg
     2641 tatttattcg gcgcaaagtg cgtcgggtga tgctgccaac ttagtcgacg gtctcttcga
     2701 actagtaagg gcgaattcga cccagctttc ttgtacaaag ttggcattat aagaaagcat
     2761 tgcttatcaa tttgttgcaa cgaacaggtc actatcagtc aaaataaaat cattatttgc
     2821 catccagctg atatccccta tagtgagtcg tattacatgg tcatagctgt ttcctggcag
     2881 ctctggcccg tgtctcaaaa tctctgatgt tacattgcac aagataaaat aatatcatca
     2941 tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt
     3001 caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat
     3061 aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgctt gtatgggaag
     3121 cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca
     3181 gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat
     3241 tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg aaaaacagca
     3301 ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg
     3361 ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta
     3421 tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt
     3481 gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg
     3541 ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt
     3601 gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac
     3661 caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg
     3721 ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg
     3781 ctcgatgagt ttttctaatc agaattggtt aattggttgt aacactggca gagcattacg
     3841 ctgacttgac gggacggcgc aagctcatga ccaaaatccc ttaacgtgag ttacgcgtcg
     3901 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     3961 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     4021 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     4081 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     4141 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     4201 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     4261 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     4321 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     4381 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     4441 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     4501 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     4561 ttcctggcct tttgctggcc ttttgctcac atgtt
//
