LOCUS       sequence-331019-        4546 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intermediate vector for two 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 4546)
  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 4546)
  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: pSF463
FEATURES             Location/Qualifiers
     source          1..4546
                     /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(1085..1390)
                     /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(1187..1206)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1710..2393)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     2308..2327
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2394..2496)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(2671..2770)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2787..2806)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2788..2806)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2811..2827)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2811..2827)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2929..2948)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2940..3749
                     /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(3007..3026)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3896..4484
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4385..4404
                     /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 caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctta tacaaaagtt gccccatggc gttccctcta gataacgcag gatccccaag
      721 tggtggctat cgagaccgtc gaccgacagc cttccaaatg ttcttctcaa acggaatcgt
      781 cgtatccagc ctactcgcta ttgtcctcaa tgccgtatta aatcataaaa agaaataaga
      841 aaaagaggtg cgagcctctt ttttgtgtga caaaataaaa acatctacct attcatatac
      901 gctagtgtca tagtcctgaa aatcatctgc atcaagaaca atttcacaac tcttatactt
      961 ttctcttaca agtcgttcgg cttcatctgg attttcagcc tctatactta ctaaacgtga
     1021 taaagtttct gtaatttcta ctgtatcgac ctgcagactg gctgtgtata agggagcctg
     1081 acatttatat tccccagaac atcaggttaa tggcgttttt gatgtcattt tcgcggtggc
     1141 tgagatcagc cacttcttcc ccgataacgg agactggcac actggccata tcggtggtca
     1201 tcatgcgcca gctttcatcc ccgatatgca ccaccgggta aagttcacgg gagactttat
     1261 ctgacagcag acgtgcactg gccaggggga tcaccatccg tcgcccgggc gtgtcaataa
     1321 tatcactctg tacatccaca aacagacgat aacggctctc tcttttatag gtgtaaacct
     1381 taaactgcat ttcaccagcc cctgttctcg tcagcaaaag agccgttcat ttcaataaac
     1441 cgggcgacct cagccatccc ttcctgattt tccgctttcc agcgttcggc acgcagacga
     1501 cgggcttcat tctgcatggt tgtgcttacc agaccggaga tattgacatc atatatgcct
     1561 tgagcaactg atagctgtcg ctgtcaactg tcactgtaat acgctgcttc atagcatacc
     1621 tctttttgac atacttcggg tatacatatc agtatatatt cttataccgc aaaaatcagc
     1681 gcgcaaatac gcatactgtt atctggcttt tagtaagccg gatccacgcg gcgtttacgc
     1741 cccccctgcc actcatcgca gtactgttgt aattcattaa gcattctgcc gacatggaag
     1801 ccatcacaaa cggcatgatg aacctgaatc gccagcggca tcagcacctt gtcgccttgc
     1861 gtataatatt tgcccatggt gaaaacgggg gcgaagaagt tgtccatatt ggccacgttt
     1921 aaatcaaaac tggtgaaact cacccaggga ttggctgaga cgaaaaacat attctcaata
     1981 aaccctttag ggaaataggc caggttttca ccgtaacacg ccacatcttg cgaatatatg
     2041 tgtagaaact gccggaaatc gtcgtggtat tcactccaga gcgatgaaaa cgtttcagtt
     2101 tgctcatgga aaacggtgta acaagggtga acactatccc atatcaccag ctcaccgtct
     2161 ttcattgcca tacggaattc cggatgagca ttcatcaggc gggcaagaat gtgaataaag
     2221 gccggataaa acttgtgctt atttttcttt acggtcttta aaaaggccgt aatatccagc
     2281 tgaacggtct ggttataggt acattgagca actgactgaa atgcctcaaa atgttcttta
     2341 cgatgccatt gggatatatc aacggtggta tatccagtga tttttttctc cattttagct
     2401 tccttagctc ctgaaaatct cgataactca aaaaatacgc ccggtagtga tcttatttca
     2461 ttatggtgaa agttggaacc tcttacgtgc cgatcaacgt ctcattttcg ccaaaagttg
     2521 gcccagggct tcccggtatc aacagggaca ccaggattta tttattctgc gaagtgatct
     2581 tccgtcacag gtatttattc ggcgcaaagt gcgtcgggtg atgctgccaa cttagtcgac
     2641 ggtctctgga cactagtaag ggtgaattcg acccagcttt cttgtacaaa gttggcatta
     2701 taagaaagca ttgcttatca atttgttgca acgaacaggt cactatcagt caaaataaaa
     2761 tcattatttg ccatccagct gatatcccct atagtgagtc gtattacatg gtcatagctg
     2821 tttcctggca gctctggccc gtgtctcaaa atctctgatg ttacattgca caagataaaa
     2881 atatatcatc atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta
     2941 tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt
     3001 tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgct
     3061 tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca
     3121 atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga
     3181 ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg
     3241 gaaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg
     3301 cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca
     3361 gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg
     3421 cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc
     3481 ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata
     3541 accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg
     3601 cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat
     3661 tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt
     3721 ttcatttgat gctcgatgag tttttctaat cagaattggt taattggttg taacactggc
     3781 agagcattac gctgacttga cgggacggcg caagctcatg accaaaatcc cttaacgtga
     3841 gttacgcgtc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3901 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     3961 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4021 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     4081 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4141 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4201 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4261 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     4321 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     4381 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     4441 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     4501 cctttttacg gttcctggcc ttttgctggc cttttgctca catgtt
//
