LOCUS       sequence-339281-        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: pGRNA3e
FEATURES             Location/Qualifiers
     source          1..3425
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..22
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(37..42,43..60,61..67)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    75..91
                     /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     80..102
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     99..115
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     99..115
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             707..1501
                     /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             707..1501
                     /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(761..780)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1371..1390
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(1519..1587,1588..2379)
                     /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(1737..1756)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2550..3138
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3039..3058
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
       61 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
      121 ttacgccaag cttcacctgc ggctggctaa caaagcacca gtggtctagt ggtagaatag
      181 taccctgcca cggtacagac ccgggttcga ttcccggctg gtgcaagaga cctccggagt
      241 ggtctcagtt tgaaagcagg tgctcgaggg gcccgttatc ggggaagaag tggctgatct
      301 cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataaat
      361 gtcaggcatg agattatcaa aaaggatctt cacctagatc cttttcacgt agaaagccag
      421 tccgcagaaa cggtgctgac cccggatgaa tgtcagctac tgggctatct ggacaaggga
      481 aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg cttacatggc gatagctaga
      541 ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc cctctggtaa
      601 ggttgggaag ccctgcaaag taaactggat ggctttcttg ccgccaagga tctgatggcg
      661 caggggatca agctctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
      721 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
      781 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
      841 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc
      901 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
      961 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     1021 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     1081 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     1141 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     1201 cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt
     1261 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     1321 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     1381 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     1441 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     1501 aattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     1561 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     1621 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     1681 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     1741 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     1801 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     1861 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     1921 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     1981 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     2041 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     2101 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     2161 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     2221 gccggtgagc gtggttcgcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     2281 cgtatcgtag ttatctacac gacggggagc caggcaacta tggatgaacg aaatagacag
     2341 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     2401 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     2461 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     2521 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     2581 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     2641 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     2701 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     2761 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     2821 ttggattcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     2881 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     2941 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     3001 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     3061 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     3121 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     3181 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     3241 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgaatca
     3301 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     3361 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     3421 gcaat
//
