LOCUS       sequence-337488-        4594 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 4594)
  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 4594)
  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..4594
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(59..145)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     194..216
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     208..225
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     209..225
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    241..340
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(805..1110)
                     /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(907..926)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1430..2113)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     2028..2047
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2114..2216)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(2391..2490)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2507..2526)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2508..2526)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2531..2547)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2531..2547)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2649..2668)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2660..3469
                     /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(2727..2746)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3616..4204
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4105..4124
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4358..4375
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4534..4561)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gtcctgcccg ccaccctccg ggccgttgct tcacaacgtt caaatccgct cccggcggat
       61 ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt
      121 ccgactgagc ctttcgtttt atttgatgcc tggcagttcc ctactctcgc gttaacgcta
      181 gcatggatgt tttcccagtc acgacgttgt aaaacgacgg ccagtcttaa gctcgggccc
      241 caaataatga ttttattttg actgatagtg acctgttcgt tgcaacacat tgatgagcaa
      301 tgctttttta taatgccaac tttgtacaaa aaagcaggct tatacaaaag ttgccccatg
      361 gcgttccctc tagataacgc aggatcccca agtggtggct atcgagaccg tcgaccgaca
      421 gccttccaaa tgttcttcgg gtgatgctgc caacttagtc gaccgacagc cttccaaatg
      481 ttcttctcaa acggaatcgt cgtatccagc ctactcgcta ttgtcctcaa tgccgtatta
      541 aatcataaaa agaaataaga aaaagaggtg cgagcctctt ttttgtgtga caaaataaaa
      601 acatctacct attcatatac gctagtgtca tagtcctgaa aatcatctgc atcaagaaca
      661 atttcacaac tcttatactt ttctcttaca agtcgttcgg cttcatctgg attttcagcc
      721 tctatactta ctaaacgtga taaagtttct gtaatttcta ctgtatcgac ctgcagactg
      781 gctgtgtata agggagcctg acatttatat tccccagaac atcaggttaa tggcgttttt
      841 gatgtcattt tcgcggtggc tgagatcagc cacttcttcc ccgataacgg agaccggcac
      901 actggccata tcggtggtca tcatgcgcca gctttcatcc ccgatatgca ccaccgggta
      961 aagttcacgg gagactttat ctgacagcag acgtgcactg gccaggggga tcaccatccg
     1021 tcgcccgggc gtgtcaataa tatcactctg tacatccaca aacagacgat aacggctctc
     1081 tcttttatag gtgtaaacct taaactgcat ttcaccagcc cctgttctcg tcagcaaaag
     1141 agccgttcat ttcaataaac cgggcgacct cagccatccc ttcctgattt tccgctttcc
     1201 agcgttcggc acgcagacga cgggcttcat tctgcatggt tgtgcttacc agaccggaga
     1261 tattgacatc atatatgcct tgagcaactg atagctgtcg ctgtcaacgg tcactgtaat
     1321 acgctgcttc atagcatacc tctttttgac atacttcggg tatacatatc agtatatatt
     1381 cttataccgc aaaaatcagc gcgcaaatac gcatactgtt atctggcttt tagtaagccg
     1441 gatccacgcg gcgtttacgc cccccctgcc actcatcgca gtactgttgt aattcattaa
     1501 gcattctgcc gacatggaag ccatcacaaa cggcatgatg aacctgaatc gccagcggca
     1561 tcagcacctt gtcgccttgc gtataatatt tgcccatggt gaaaacgggg gcgaagaagt
     1621 tgtccatatt ggccacgttt aaatcaaaac tggtgaaact cacccaggga ttggctgaga
     1681 cgaaaaacat attctcaata aaccctttag ggaaataggc caggttttca ccgtaacacg
     1741 ccacatcttg cgaatatatg tgtagaaact gccggaaatc gtcgtggtat tcactccaga
     1801 gcgatgaaaa cgtttcagtt tgctcatgga aaacggtgta acaagggtga acactatccc
     1861 atatcaccag ctcaccgtct ttcattgcca tacggaattc cggatgagca ttcatcaggc
     1921 gggcaagaat gtgaataaag gccggataaa acttgtgctt atttttcttt acggtcttta
     1981 aaaaggccgt aatatccagc tgaacggtct ggttataggt acattgagca actgactgaa
     2041 atgcctcaaa atgttcttta cgatgccatt gggatatatc aacggtggta tatccagtga
     2101 tttttttctc cattttagct tccttagctc ctgaaaatct cgataactca aaaaatacgc
     2161 ccggtagtga tcttatttca ttatggtgaa agttggaacc tcttacgtgc cgatcaacgt
     2221 ctcattttcg ccaaaagttg gcccagggct tcccggtatc aacagggaca ccaggattta
     2281 tttattctgc gaagtgatct tccgtcacag gtatttattc ggcgcaaagt gcgtcgggtg
     2341 atgctgccaa cttagtcgac ggtctcttcg aactagtaag ggcgaattcg acccagcttt
     2401 cttgtacaaa gttggcatta taagaaagca ttgcttatca atttgttgca acgaacaggt
     2461 cactatcagt caaaataaaa tcattatttg ccatccagct gatatcccct atagtgagtc
     2521 gtattacatg gtcatagctg tttcctggca gctctggccc gtgtctcaaa atctctgatg
     2581 ttacattgca caagataaaa atatatcatc atgaacaata aaactgtctg cttacataaa
     2641 cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa
     2701 ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc
     2761 aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca
     2821 tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac
     2881 ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt
     2941 actcaccact gcgatccccg gaaaaacagc attccaggta ttagaagaat atcctgattc
     3001 aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt
     3061 ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat
     3121 gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga
     3181 acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca
     3241 tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga
     3301 tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct
     3361 cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc
     3421 tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat cagaattggt
     3481 taattggttg taacactggc agagcattac gctgacttga cgggacggcg caagctcatg
     3541 accaaaatcc cttaacgtga gttacgcgtc gttccactga gcgtcagacc ccgtagaaaa
     3601 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     3661 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     3721 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     3781 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     3841 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     3901 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     3961 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     4021 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     4081 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     4141 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     4201 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     4261 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     4321 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     4381 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     4441 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatacgcg
     4501 taccgctagc caggaagagt ttgtagaaac gcaaaaaggc catccgtcag gatggccttc
     4561 tgcttagttt gatgcctggc agtttatggc gggc
//
