LOCUS       sequence-335586-        3877 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ENTRY vector for gRNA4 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: pRU44
FEATURES             Location/Qualifiers
     source          1..3877
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(24..41)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(195..783)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(275..294)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(954..1745,1746..1814))
                     /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     1577..1596
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1815..1919)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1997..2452)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2134..2155)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2346..2365
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2578..2600
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2592..2609
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2593..2609
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2616..2635
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2616..2634
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2665..2674
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2669..2678
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_RNA        complement(3064..3139)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(3634..3652)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3635..3652)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3670..3686)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3670..3686)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3683..3705)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3694..3710
                     /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(3718..3724,3725..3742,3743..3748))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3763..3784
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
       61 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
      121 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
      181 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
      241 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
      301 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
      361 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
      421 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
      481 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
      541 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
      601 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
      661 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
      721 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
      781 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
      841 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
      901 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
      961 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     1021 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     1081 gcaatgatac cgcgagatcc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     1141 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     1201 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     1261 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     1321 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     1381 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     1441 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     1501 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     1561 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     1621 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     1681 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     1741 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     1801 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     1861 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     1921 acatttcccc gaaaagtgcc acctgatgcg gtgtgaaata ccgcacagat gcgtaaggag
     1981 aaaataccgc atcatgaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     2041 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     2101 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     2161 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     2221 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     2281 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     2341 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     2401 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtccattcgc
     2461 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     2521 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
     2581 agtcacgacg ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat
     2641 tgggcccgac gtcgcatgct cccggccgcc atggcggccg gtctccaaca gctgatccta
     2701 aatgctatca agtttattat atttttgtga aagaaatgtt gtgtttcaat ctttggtcta
     2761 tctaatttca cccttcataa gaaaatgatg tagccgtctg gaaacaaact ctcgatacat
     2821 ttcttacccg aaatctggtt aacaaaacgc attggtgtcg ccgactcgaa acaaactctc
     2881 tgacaatcag aactgaaaca acggaccaat cactttgtct tagctatcta aataagctca
     2941 ttgcaggtcc aacttttttc gcgcagattt gcatccaaaa cttcacttat gattcacgaa
     3001 acggaaaaaa aaacccagaa atctacagat gaagaaaaaa tctgaaaatt tttgccaaaa
     3061 aaagcaccga ctcggtgcca ctttttcaag ttgataacgg actagcctta ttttaacttg
     3121 ctatttctag ctctaaaaca ggtcttctcg aagaccctga ccaatggtgc tccctcagtg
     3181 ttatatatac aacaagattt gtttagattt gagcgatgtg ggagaaatta ggagcttata
     3241 tgaaacgcat acatgggtac tgggcatggg tattagtgaa tatatggacg atagcccatt
     3301 aaattacata aaaagcccac ttatcttatt gctcgatata agctccgtat ataaacggaa
     3361 gttgatccta aaagttcact tgtttcgtcc aacatagcca aaacagatgt tcttaaaccc
     3421 agacgccatt tgtttgattc agttatccat cacaggctga gccataagaa aaatttaaag
     3481 taaaatagcg acacctctag aggaggcatg atgatatatt tttatcttgt gcaatgtaac
     3541 atcagagatt ttgagacacg ctggcgagac cggccgcctg caggtcgacc atatgggaga
     3601 gctcccaacg cgttggatgc atagcttgag tattctatag tgtcacctaa atagcttggc
     3661 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     3721 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     3781 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     3841 ttaatgaatc ggccaacgcg cggggagagg cggtttg
//
