LOCUS       sequence-331846-        3864 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ENTRY vector for gRNA3 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: pRU43
FEATURES             Location/Qualifiers
     source          1..3864
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(11..28)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(182..770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(262..281)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(941..1732,1733..1801))
                     /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     1564..1583
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1802..1906)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1984..2439)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2121..2142)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2333..2352
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2565..2587
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2579..2596
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2580..2596
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2603..2622
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2603..2621
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2652..2661
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2656..2665
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_RNA        complement(3051..3126)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(3608..3626)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3609..3626)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3644..3660)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3644..3660)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3657..3679)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3668..3684
                     /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(3692..3698,3699..3716,3717..3722))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3737..3758
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
       61 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
      121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
      181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      601 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
      661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
      841 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
      901 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
      961 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     1021 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     1081 gagatccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     1141 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     1201 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     1261 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     1321 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     1381 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     1441 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     1501 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     1561 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     1621 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     1681 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     1741 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     1801 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     1861 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     1921 aagtgccacc tgatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     1981 aggaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     2041 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     2101 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     2161 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     2221 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     2281 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     2341 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     2401 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc cattcgccat tcaggctgcg
     2461 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
     2521 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
     2581 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg gcccgacgtc
     2641 gcatgctccc ggccgccatg gcggccggtc tccctgggct gatcctaaat gctatcaagt
     2701 ttattatatt tttgtgaaag aaatgttgtg tttcaatctt tggtctatct aatttcaccc
     2761 ttcataagaa aatgatgtag ccgtctggaa acaaactctc gatacatttc ttacccgaaa
     2821 tctggttaac aaaacgcatt ggtgtcgccg actcgaaaca aactctctga caatcagaac
     2881 tgaaacaacg gaccaatcac tttgtcttag ctatctaaat aagctcattg caggtccaac
     2941 ttttttcgcg cagatttgca tccaaaactt cacttatgat tcacgaaacg gaaaaaaaaa
     3001 cccagaaatc tacagatgaa gaaaaaatct gaaaattttt gccaaaaaaa gcaccgactc
     3061 ggtgccactt tttcaagttg ataacggact agccttattt taacttgcta tttctagctc
     3121 taaaacaggt cttctcgaag accccaatca ctacttcgac tctagctgta tataaactca
     3181 gcttcgtttt cttatctaag cgatgtggga cttttgaaga ttgttttcaa cttaaatggg
     3241 cctatataag aaatactatt gttctttccc atataaatgg gcctgcttct cttctttcag
     3301 attcccaggg gccttttgaa gattatcttc atatcttaag aatgaagatg ttttattcaa
     3361 tcaaattctt gaaggttcga tgcctaatca ttctaatcct gggacaaact atgaaacaag
     3421 atacaaaaac tccgaatgga aagttaaaaa gaagaaaacg aaagctacgg ttcaagaaaa
     3481 tgtaagctga taaacaaaaa aaaactgtat gaacgaagaa gaagaaaaaa aggtcctgag
     3541 accgcggccg cctgcaggtc gaccatatgg gagagctccc aacgcgttgg atgcatagct
     3601 tgagtattct atagtgtcac ctaaatagct tggcgtaatc atggtcatag ctgtttcctg
     3661 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     3721 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     3781 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     3841 gaggcggttt gcgtattggg cgct
//
