LOCUS       sequence-417710-        3871 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ENTRY vector for gRNA5 cloning under pAtU6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3871)
  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 3871)
  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: pRU45
FEATURES             Location/Qualifiers
     source          1..3871
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(5..24)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6..24)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(31..48)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(31..47)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(40..62)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      188..643
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(275..294)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     485..506
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        721..825
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(826..894,895..1686)
                     /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(1044..1063)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1857..2445
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2346..2365
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2599..2616
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2733..2754
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2769..2774,2775..2792,2793..2799)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2807..2823
                     /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     2812..2834
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2831..2847
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2831..2847
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2865..2883
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2865..2882
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_RNA        3367..3442
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      3828..3837
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      3832..3841
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 ttcgccctat agtgagtcgt attacaattc actggccgtc gttttacaac gtcgtgactg
       61 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
      121 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
      181 cgaatggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
      241 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
      301 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
      361 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
      421 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
      481 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
      541 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
      601 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttcctgatg cggtattttc
      661 tccttacgca tctgtgcggt atttcacacc gcatcaggtg gcacttttcg gggaaatgtg
      721 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
      781 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
      841 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
      901 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
      961 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     1021 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     1081 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     1141 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     1201 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     1261 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     1321 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     1381 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     1441 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     1501 ggctggttta ttgctgataa atctggagcc ggtgagcgtg gatctcgcgg tatcattgca
     1561 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     1621 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     1681 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     1741 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     1801 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     1861 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     1921 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     1981 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     2041 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     2101 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     2161 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     2221 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     2281 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     2341 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     2401 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     2461 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     2521 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     2581 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
     2641 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     2701 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
     2761 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
     2821 caatttcaca caggaaacag ctatgaccat gattacgcca agctatttag gtgacactat
     2881 agaatactca agctatgcat ccaacgcgtt gggagctctc ccatatgtat ggtcgacctg
     2941 caggcggccg gtctcctgtt ctttttttct tcttcttcgt tcatacagtt tttttttgtt
     3001 tatcagctta cattttcttg aaccgtagct ttcgttttct tctttttaac tttccattcg
     3061 gagtttttgt atcttgtttc atagtttgtc ccaggattag aatgattagg catcgaacct
     3121 tcaagaattt gattgaataa aacatcttca ttcttaagat atgaagataa tcttcaaaag
     3181 gcccctggga atctgaaaga agagaagcag gcccatttat atgggaaaga acaatagtat
     3241 ttcttatata ggcccattta agttgaaaac aatcttcaaa agtcccacat cgcttagata
     3301 agaaaacgaa gctgagttta tatacagcta gagtcgaagt agtgattggg gtcttcgaga
     3361 agacctgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
     3421 aaaagtggca ccgagtcggt gctttttttg gcaaaaattt tcagattttt tcttcatctg
     3481 tagatttctg ggtttttttt tccgtttcgt gaatcataag tgaagttttg gatgcaaatc
     3541 tgcgcgaaaa aagttggacc tgcaatgagc ttatttagat agctaagaca aagtgattgg
     3601 tccgttgttt cagttctgat tgtcagagag tttgtttcga gtcggcgaca ccaatgcgtt
     3661 ttgttaacca gatttcgggt aagaaatgta tcgagagttt gtttccagac ggctacatca
     3721 ttttcttatg aagggtgaaa ttagatagac caaagattga aacacaacat ttctttcaca
     3781 aaaatataat aaacttgata gcatttagga tcagctgcag gagaccggcc gccatggcgg
     3841 ccgggagcat gcgacgtcgg gccgggccca a
//
