LOCUS       sequence-338358-        3425 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Insert one gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3425)
  AUTHORS   Oh Y, Lee B, Kim H, Kim SG
  TITLE     A multiplex guide RNA expression system and its efficacy for plant 
            genome engineering.
  JOURNAL   Plant Methods. 2020 Mar 12;16:37. doi: 10.1186/s13007-020-00580-x. 
            eCollection 2020.
  PUBMED    32190101
REFERENCE   2  (bases 1 to 3425)
  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: "2020-03-12"; volume: "16:37. doi"; pages: " 
            10.1186/s13007-020-00580-x. eCollection 2020"
COMMENT     Sequence Label: pGRNA5e
FEATURES             Location/Qualifiers
     source          1..3425
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(255..271)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(255..271)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(268..290)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    279..295
                     /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(303..309,310..327,328..333))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    348..369
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(657..1245)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(737..756)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1416..2207,2208..2276))
                     /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     2039..2058
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             complement(2294..3088)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(2294..3088)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2405..2424)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3015..3034
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 atgcctgaca tttatattcc ccagaacatc aggttaatgg cgtttttgat gtcattttcg
       61 cggtggctga gatcagccac ttcttccccg ataacgggcc cctcgagcac ctgctttcaa
      121 actgagacca ctccggaggt ctcttgcacc agccgggaat cgaacccggg tctgtaccgt
      181 ggcagggtac tattctacca ctagaccact ggtgctttgt tagctagctg caggtgaagc
      241 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
      301 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
      361 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
      421 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
      481 gcttcctcgc tcactgattc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
      541 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
      601 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
      661 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
      721 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
      781 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
      841 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
      901 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
      961 cgtcttgaat ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     1021 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     1081 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     1141 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     1201 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     1261 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     1321 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     1381 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     1441 cctatctcag cgatctgtct atttcgttca tccatagttg cctggctccc cgtcgtgtag
     1501 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgcgaa
     1561 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     1621 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     1681 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     1741 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     1801 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     1861 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     1921 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     1981 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     2041 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     2101 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     2161 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     2221 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     2281 ttcctttttc aattcagaag aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat
     2341 cgggagcggc gataccgtaa agcacgagga agcggtcagc ccattcgccg ccaagctctt
     2401 cagcaatatc acgggtagcc aacgctatgt cctgatagcg gtccgccaca cccagccggc
     2461 cacagtcgat gaatccagaa aagcggccat tttccaccat gatattcggc aagcaggcat
     2521 cgccatgggt cacgacgaga tcctcgccgt cgggcatgct cgccttgagc ctggcgaaca
     2581 gttcggctgg cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg
     2641 cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg
     2701 tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg
     2761 caggagcaag gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt
     2821 cccttcccgc ttcagtgaca acgtcgagca cagctgcgca aggaacgccc gtcgtggcca
     2881 gccacgatag ccgcgctgcc tcgtcttgca gttcattcag ggcaccggac aggtcggtct
     2941 tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc
     3001 cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg gccggagaac
     3061 ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc tcttgatcag
     3121 agcttgatcc cctgcgccat cagatccttg gcggcaagaa agccatccag tttactttgc
     3181 agggcttccc aaccttacca gagggcgccc cagctggcaa ttccggttcg cttgctgtcc
     3241 ataaaaccgc ccagtctagc tatcgccatg taagcccact gcaagctacc tgctttctct
     3301 ttgcgcttgc gttttccctt gtccagatag cccagtagct gacattcatc cggggtcagc
     3361 accgtttctg cggactggct ttctacgtga aaaggatcta ggtgaagatc ctttttgata
     3421 atctc
//
