LOCUS       sequence-338356-        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: pGRNA4e
FEATURES             Location/Qualifiers
     source          1..3425
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    129..150
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(165..170,171..188,189..195)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    203..219
                     /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     208..230
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     227..243
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     227..243
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             835..1629
                     /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             835..1629
                     /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(889..908)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1499..1518
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(1647..1715,1716..2507)
                     /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(1865..1884)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2678..3266
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3167..3186
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg
       61 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
      121 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
      181 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
      241 gaccatgatt acgccaagct tcacctgcga aggaagaaca aagcaccagt ggtctagtgg
      301 tagaatagta ccctgccacg gtacagaccc gggttcgatt cccggctggt gcaagagacc
      361 tccggagtgg tctcagtttg aaagcaggtg ctcgaggggc ccgttatcgg ggaagaagtg
      421 gctgatctca gccaccgcga aaatgacatc aaaaacgcca ttaacctgat gttctgggga
      481 atataaatgt caggcatgag attatcaaaa aggatcttca cctagatcct tttcacgtag
      541 aaagccagtc cgcagaaacg gtgctgaccc cggatgaatg tcagctactg ggctatctgg
      601 acaagggaaa acgcaagcgc aaagagaaag caggtagctt gcagtgggct tacatggcga
      661 tagctagact gggcggtttt atggacagca agcgaaccgg aattgccagc tggggcgccc
      721 tctggtaagg ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc gccaaggatc
      781 tgatggcgca ggggatcaag ctctgatcaa gagacaggat gaggatcgtt tcgcatgatt
      841 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
      901 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
      961 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac
     1021 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     1081 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     1141 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     1201 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     1261 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     1321 caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag
     1381 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     1441 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     1501 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     1561 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     1621 ttcttctgaa ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     1681 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     1741 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     1801 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     1861 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     1921 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     1981 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     2041 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     2101 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     2161 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     2221 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     2281 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     2341 aatctggagc cggtgagcgt ggttcgcgcg gtatcattgc agcactgggg ccagatggta
     2401 agccctcccg tatcgtagtt atctacacga cggggagcca ggcaactatg gatgaacgaa
     2461 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     2521 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     2581 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     2641 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     2701 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     2761 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     2821 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     2881 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     2941 ttaccgggtt ggattcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     3001 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     3061 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     3121 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     3181 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     3241 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     3301 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     3361 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     3421 gcgaa
//
