LOCUS       sequence-417705-        3873 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ENTRY vector for gRNA7 cloning under pAtU6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3873)
  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 3873)
  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: pRU47
FEATURES             Location/Qualifiers
     source          1..3873
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(2..21)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3..21)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(28..45)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(28..44)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(37..59)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      185..640
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     482..503
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        718..822
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(823..891,892..1683)
                     /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(1041..1060)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1854..2442
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2343..2362
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2596..2613
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2730..2751
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2766..2771,2772..2789,2790..2796)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2804..2820
                     /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     2809..2831
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2828..2844
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2828..2844
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2862..2880
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2862..2879
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_RNA        3366..3441
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      3827..3836
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      3831..3840
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 gccctatagt gagtcgtatt acaattcact ggccgtcgtt ttacaacgtc gtgactggga
       61 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
      121 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
      181 atggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
      241 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
      301 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
      361 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
      421 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
      481 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
      541 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
      601 tttaacgcga attttaacaa aatattaacg cttacaattt cctgatgcgg tattttctcc
      661 ttacgcatct gtgcggtatt tcacaccgca tcaggtggca cttttcgggg aaatgtgcgc
      721 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
      781 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
      841 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
      901 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
      961 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     1021 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     1081 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     1141 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     1201 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     1261 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     1321 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     1381 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     1441 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     1501 tggtttattg ctgataaatc tggagccggt gagcgtggat ctcgcggtat cattgcagca
     1561 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     1621 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     1681 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     1741 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     1801 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     1861 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     1921 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     1981 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     2041 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     2101 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     2161 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     2221 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     2281 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     2341 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     2401 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     2461 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     2521 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     2581 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
     2641 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     2701 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
     2761 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
     2821 tttcacacag gaaacagcta tgaccatgat tacgccaagc tatttaggtg acactataga
     2881 atactcaagc tatgcatcca acgcgttggg agctctccca tatgtatggt cgacctgcag
     2941 gcggccgcgg tctcgccggc tttttttctt cttcttcgtt catacagttt ttttttgttt
     3001 atcagcttac attttcttga accgtagctt tcgttttctt ctttttaact ttccattcgg
     3061 agtttttgta tcttgtttca tagtttgtcc caggattaga atgattaggc atcgaacctt
     3121 caagaatttg attgaataaa acatcttcat tcttaagata tgaagataat cttcaaaagg
     3181 cccctgggaa tctgaaagaa gagaagcagg cccatttata tgggaaagaa caatagtatt
     3241 tcttatatag gcccatttaa gttgaaaaca atcttcaaaa gtcccacatc gcttagataa
     3301 gaaaacgaag ctgagtttat atacagctag agtcgaagta gtgattgggg tcttcgagaa
     3361 gacctgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa
     3421 aaagtggcac cgagtcggtg ctttttttgg caaaaatttt cagatttttt cttcatctgt
     3481 agatttctgg gttttttttt ccgtttcgtg aatcataagt gaagttttgg atgcaaatct
     3541 gcgcgaaaaa agttggacct gcaatgagct tatttagata gctaagacaa agtgattggt
     3601 ccgttgtttc agttctgatt gtcagagagt ttgtttcgag tcggcgacac caatgcgttt
     3661 tgttaaccag atttcgggta agaaatgtat cgagagtttg tttccagacg gctacatcat
     3721 tttcttatga agggtgaaat tagatagacc aaagattgaa acacaacatt tctttcacaa
     3781 aaatataata aacttgatag catttaggat cagcgaaagg agaccggccg ccatggcggc
     3841 cgggagcatg cgacgtcggg ccgggcccaa ttc
//
