LOCUS       sequence-331965-        3876 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ENTRY vector for gRNA6 cloning under pAtU3 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3876)
  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 3876)
  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: pRU46
FEATURES             Location/Qualifiers
     source          1..3876
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      92..680
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     581..600
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     834..851
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    968..989
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1004..1009,1010..1027,1028..1034)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1042..1058
                     /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     1047..1069
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1066..1082
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1066..1082
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1100..1118
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1100..1117
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_RNA        1613..1688
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      2073..2082
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2077..2086
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2116..2135)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2117..2135)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2142..2159)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2142..2158)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2151..2173)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2299..2754
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2386..2405)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2596..2617
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2832..2936
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2937..3005,3006..3797)
                     /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(3155..3174)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
       61 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
      121 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
      181 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
      241 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
      301 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
      361 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
      421 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
      481 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
      541 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
      601 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
      661 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
      721 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
      781 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
      841 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
      901 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
      961 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     1021 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     1081 accatgatta cgccaagcta tttaggtgac actatagaat actcaagcta tgcatccaac
     1141 gcgttgggag ctctcccata tggtcgacct gcaggcggcc ggtctcgtgc acgtgtctca
     1201 aaatctctga tgttacattg cacaagataa aaatatatca tcatgcctcc tctagaggtg
     1261 tcgctatttt actttaaatt tttcttatgg ctcagcctgt gatggataac tgaatcaaac
     1321 aaatggcgtc tgggtttaag aacatctgtt ttggctatgt tggacgaaac aagtgaactt
     1381 ttaggatcaa cttccgttta tatacggagc ttatatcgag caataagata agtgggcttt
     1441 ttatgtaatt taatgggcta tcgtccatat attcactaat acccatgccc agtacccatg
     1501 tatgcgtttc atataagctc ctaatttctc ccacatcgct caaatctaaa caaatcttgt
     1561 tgtatatata acactgaggg agcaccattg gtcagggtct tcgagaagac ctgttttaga
     1621 gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga
     1681 gtcggtgctt tttttggcaa aaattttcag attttttctt catctgtaga tttctgggtt
     1741 tttttttccg tttcgtgaat cataagtgaa gttttggatg caaatctgcg cgaaaaaagt
     1801 tggacctgca atgagcttat ttagatagct aagacaaagt gattggtccg ttgtttcagt
     1861 tctgattgtc agagagtttg tttcgagtcg gcgacaccaa tgcgttttgt taaccagatt
     1921 tcgggtaaga aatgtatcga gagtttgttt ccagacggct acatcatttt cttatgaagg
     1981 gtgaaattag atagaccaaa gattgaaaca caacatttct ttcacaaaaa tataataaac
     2041 ttgatagcat ttaggatcag ccggtgagac cggccgccat ggcggccggg agcatgcgac
     2101 gtcgggccca attcgcccta tagtgagtcg tattacaatt cactggccgt cgttttacaa
     2161 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     2221 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     2281 agcctgaatg gcgaatggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     2341 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     2401 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     2461 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     2521 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     2581 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     2641 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     2701 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttcctgat
     2761 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatcaggt ggcacttttc
     2821 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     2881 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     2941 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     3001 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     3061 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     3121 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     3181 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     3241 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     3301 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     3361 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     3421 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     3481 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     3541 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     3601 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt ggatctcgcg
     3661 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     3721 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     3781 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     3841 aacttcattt ttaatttaaa aggatctagg tgaaga
//
