LOCUS       sequence-331852-        3877 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ENTRY vector for gRNA8 cloning under pAtU3 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3877)
  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 3877)
  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: pRU48
FEATURES             Location/Qualifiers
     source          1..3877
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(16..33)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(187..775)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(267..286)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(946..1737,1738..1806))
                     /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     1569..1588
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1807..1911)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1989..2444)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2126..2147)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2338..2357
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2570..2592
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2584..2601
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2585..2601
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2608..2627
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2608..2626
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2657..2666
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2661..2670
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_RNA        complement(3056..3131)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(3626..3644)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3627..3644)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3662..3678)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3662..3678)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3675..3697)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3686..3702
                     /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)."
     promoter        complement(join(3710..3716,3717..3734,3735..3740))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3755..3776
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
       61 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
      121 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
      181 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
      241 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
      301 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
      361 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
      421 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
      481 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
      541 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
      601 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
      661 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
      721 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
      781 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
      841 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
      901 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
      961 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     1021 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     1081 accgcgagat ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     1141 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     1201 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     1261 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     1321 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     1381 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     1441 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     1501 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     1561 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     1621 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     1681 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     1741 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     1801 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     1861 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     1921 ccgaaaagtg ccacctgatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
     1981 gcatcatgaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     2041 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     2101 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     2161 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     2221 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     2281 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     2341 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     2401 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtccattc gccattcagg
     2461 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg
     2521 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
     2581 cgttgtaaaa cgacggccag tgaattgtaa tacgactcac tatagggcga attgggcccg
     2641 acgtcgcatg ctcccggccg ccatggcggc cggtctcgtc gagctgatcc taaatgctat
     2701 caagtttatt atatttttgt gaaagaaatg ttgtgtttca atctttggtc tatctaattt
     2761 cacccttcat aagaaaatga tgtagccgtc tggaaacaaa ctctcgatac atttcttacc
     2821 cgaaatctgg ttaacaaaac gcattggtgt cgccgactcg aaacaaactc tctgacaatc
     2881 agaactgaaa caacggacca atcactttgt cttagctatc taaataagct cattgcaggt
     2941 ccaacttttt tcgcgcagat ttgcatccaa aacttcactt atgattcacg aaacggaaaa
     3001 aaaaacccag aaatctacag atgaagaaaa aatctgaaaa tttttgccaa aaaaagcacc
     3061 gactcggtgc cactttttca agttgataac ggactagcct tattttaact tgctatttct
     3121 agctctaaaa caggtcttct cgaagaccct gaccaatggt gctccctcag tgttatatat
     3181 acaacaagat ttgtttagat ttgagcgatg tgggagaaat taggagctta tatgaaacgc
     3241 atacatgggt actgggcatg ggtattagtg aatatatgga cgatagccca ttaaattaca
     3301 taaaaagccc acttatctta ttgctcgata taagctccgt atataaacgg aagttgatcc
     3361 taaaagttca cttgtttcgt ccaacatagc caaaacagat gttcttaaac ccagacgcca
     3421 tttgtttgat tcagttatcc atcacaggct gagccataag aaaaatttaa agtaaaatag
     3481 cgacacctct agaggaggca tgatgatata tttttatctt gtgcaatgta acatcagaga
     3541 ttttgagaca cgtttcggag accggccgcc tgcaggtcga ccatatggga gagctcccaa
     3601 cgcgttggat gcatagcttg agtattctat agtgtcacct aaatagcttg gcgtaatcat
     3661 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     3721 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     3781 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     3841 tcggccaacg cgcggggaga ggcggtttgc gtattgg
//
