LOCUS       sequence-331102-        3864 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ENTRY vector for gRNA1 cloning under pAtU6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3864)
  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 3864)
  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: pRU41
FEATURES             Location/Qualifiers
     source          1..3864
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(24..41)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(24..40)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(33..55)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      181..636
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(268..287)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     478..499
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        714..818
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(819..887,888..1679)
                     /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     complement(1037..1056)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1850..2438
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2339..2358
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2592..2609
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2726..2747
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2762..2767,2768..2785,2786..2792)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2800..2816
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     2805..2827
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2824..2840
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2824..2840
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2858..2876
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2858..2875
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_RNA        3358..3433
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      3819..3828
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      3823..3832
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(join(3862..3864,1..17))
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(join(3863..3864,1..17))
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 tatagtgagt cgtattacaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac
       61 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
      121 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
      181 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
      241 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
      301 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
      361 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
      421 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
      481 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
      541 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
      601 acgcgaattt taacaaaata ttaacgctta caatttcctg atgcggtatt ttctccttac
      661 gcatctgtgc ggtatttcac accgcatcag gtggcacttt tcggggaaat gtgcgcggaa
      721 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
      781 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
      841 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
      901 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
      961 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     1021 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     1081 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     1141 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     1201 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     1261 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     1321 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     1381 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     1441 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     1501 ttattgctga taaatctgga gccggtgagc gtggatctcg cggtatcatt gcagcactgg
     1561 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     1621 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     1681 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     1741 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     1801 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     1861 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     1921 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     1981 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     2041 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     2101 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     2161 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     2221 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     2281 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     2341 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     2401 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     2461 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     2521 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     2581 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     2641 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     2701 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
     2761 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
     2821 acacaggaaa cagctatgac catgattacg ccaagctatt taggtgacac tatagaatac
     2881 tcaagctatg catccaacgc gttgggagct ctcccatatg gtcgacctgc aggcggccgc
     2941 ggtctcgcta tctttttttc ttcttcttcg ttcatacagt ttttttttgt ttatcagctt
     3001 acattttctt gaaccgtagc tttcgttttc ttctttttaa ctttccattc ggagtttttg
     3061 tatcttgttt catagtttgt cccaggatta gaatgattag gcatcgaacc ttcaagaatt
     3121 tgattgaata aaacatcttc attcttaaga tatgaagata atcttcaaaa ggcccctggg
     3181 aatctgaaag aagagaagca ggcccattta tatgggaaag aacaatagta tttcttatat
     3241 aggcccattt aagttgaaaa caatcttcaa aagtcccaca tcgcttagat aagaaaacga
     3301 agctgagttt atatacagct agagtcgaag tagtgattgg ggtcttcgag aagacctgtt
     3361 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
     3421 accgagtcgg tgcttttttt ggcaaaaatt ttcagatttt ttcttcatct gtagatttct
     3481 gggttttttt ttccgtttcg tgaatcataa gtgaagtttt ggatgcaaat ctgcgcgaaa
     3541 aaagttggac ctgcaatgag cttatttaga tagctaagac aaagtgattg gtccgttgtt
     3601 tcagttctga ttgtcagaga gtttgtttcg agtcggcgac accaatgcgt tttgttaacc
     3661 agatttcggg taagaaatgt atcgagagtt tgtttccaga cggctacatc attttcttat
     3721 gaagggtgaa attagataga ccaaagattg aaacacaaca tttctttcac aaaaatataa
     3781 taaacttgat agcatttagg atcagccatg ggagaccggc cgccatggcg gccgggagca
     3841 tgcgacgtcg ggcccaattc gccc
//
