LOCUS       sequence-331044-        4594 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intermediate vector for five 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: pSF279
FEATURES             Location/Qualifiers
     source          1..4594
                     /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(1758..2441)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     2356..2375
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2442..2544)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(2719..2818)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2835..2854)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2836..2854)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2859..2875)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2859..2875)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2977..2996)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2988..3797
                     /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(3055..3074)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3944..4532
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4433..4452
                     /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 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 cccctgccac
     1801 tcatcgcagt actgttgtaa ttcattaagc attctgccga catggaagcc atcacaaacg
     1861 gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt ataatatttg
     1921 cccatggtga aaacgggggc gaagaagttg tccatattgg ccacgtttaa atcaaaactg
     1981 gtgaaactca cccagggatt ggctgagacg aaaaacatat tctcaataaa ccctttaggg
     2041 aaataggcca ggttttcacc gtaacacgcc acatcttgcg aatatatgtg tagaaactgc
     2101 cggaaatcgt cgtggtattc actccagagc gatgaaaacg tttcagtttg ctcatggaaa
     2161 acggtgtaac aagggtgaac actatcccat atcaccagct caccgtcttt cattgccata
     2221 cggaattccg gatgagcatt catcaggcgg gcaagaatgt gaataaaggc cggataaaac
     2281 ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa tatccagctg aacggtctgg
     2341 ttataggtac attgagcaac tgactgaaat gcctcaaaat gttctttacg atgccattgg
     2401 gatatatcaa cggtggtata tccagtgatt tttttctcca ttttagcttc cttagctcct
     2461 gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc ttatttcatt atggtgaaag
     2521 ttggaacctc ttacgtgccg atcaacgtct cattttcgcc aaaagttggc ccagggcttc
     2581 ccggtatcaa cagggacacc aggatttatt tattctgcga agtgatcttc cgtcacaggt
     2641 atttattcgg cgcaaagtgc gtcgggtgat gctgccaact tagtcgacgg tctcttgcaa
     2701 ctagtaaggg cgaattcgac ccagctttct tgtacaaagt tggcattata agaaagcatt
     2761 gcttatcaat ttgttgcaac gaacaggtca ctatcagtca aaataaaatc attatttgcc
     2821 atccagctga tatcccctat agtgagtcgt attacatggt catagctgtt tcctggcagc
     2881 tctggcccgt gtctcaaaat ctctgatgtt acattgcaca agataaaaat atatcatcat
     2941 gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc
     3001 aacgggaaac gtcgaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata
     3061 aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgcttg tatgggaagc
     3121 ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag
     3181 atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt
     3241 ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccgga aaaacagcat
     3301 tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt
     3361 tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat
     3421 ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg
     3481 atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc
     3541 cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg
     3601 acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc
     3661 aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc
     3721 tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc
     3781 tcgatgagtt tttctaatca gaattggtta attggttgta acactggcag agcattacgc
     3841 tgacttgacg ggacggcgca agctcatgac caaaatccct taacgtgagt tacgcgtcgt
     3901 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3961 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     4021 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     4081 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     4141 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     4201 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     4261 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     4321 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     4381 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     4441 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     4501 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     4561 tcctggcctt ttgctggcct tttgctcaca tgtt
//
